O2 affects mitochondrial functionality ex vivo.

Mitochondria have originated in eukaryotic cells by endosymbiosis of a specialized prokaryote approximately 2 billion years ago. They are essential for normal cell function by providing energy through their role in oxidizing carbon substrates. Glutathione (GSH) is a major thiol-disulfide redox buffer of the cell including the mitochondrial matrix and intermembrane space. We have generated cardiomyocyte-specific Grx1-roGFP2 GSH redox potential (EGSH) biosensor mice in the past, in which the sensor is targeted to the mitochondrial matrix. Using this mouse model a distinct EGSH of the mitochondrial matrix (-278.9 ±â€¯0.4 mV) in isolated cardiomyocytes is observed. When analyzing the EGSH in isolated mitochondria from the transgenic hearts, however, the EGSH in the mitochondrial matrix is significantly oxidized (-247.7 ±â€¯8.7 mV). This is prevented by adding N-Ethylmaleimide during the mitochondria isolation procedure, which precludes disulfide bond formation. A similar reducing effect is observed by isolating mitochondria in hypoxic (0.1-3% O2) conditions that mimics mitochondrial pO2 levels in cellulo. The reduced EGSH is accompanied by lower ROS production, reduced complex III activity but increased ATP levels produced at baseline and after stimulation with succinate/ADP. Altogether, we demonstrate that oxygenation is an essential factor that needs to be considered when analyzing mitochondrial function ex vivo.

Transgenic Organisms Meet Redox Bioimaging: One Step Closer to Physiology.

SIGNIFICANCE: Redox signaling is a common mechanism in the cellular response toward a variety of stimuli. For analyzing redox-dependent specific alterations in a cell, genetically encoded biosensors were highly instrumental in the past. To advance the knowledge about the importance of this signaling mechanism in vivo, models that are as close as possible to physiology are needed. Recent Advances: The development of transgenic (tg) redox biosensor animal models has enhanced the knowledge of redox signaling under patho(physio)logical conditions. So far, commonly used small animal models, that is, Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio, and genetically modified mice were employed for redox biosensor transgenesis. However, especially the available mouse models are still limited. CRITICAL ISSUES: The analysis of redox biosensor responses in vivo at the tissue level, especially for internal organs, is hampered by the detection limit of the available redox biosensors and microscopy techniques. Recent technical developments such as redox histology and the analysis of cell-type-specific biosensor responses need to be further refined and followed up in a systematic manner. FUTURE DIRECTIONS: The usage of tg animal models in the field of redox signaling has helped to answer open questions. Application of the already established models and consequent development of more defined tg models will enable this research area to define the role of redox signaling in (patho)physiology in further depth. Antioxid. Redox Signal. 29, 603-612.

X-ray-based lung function measurement reveals persistent loss of lung tissue elasticity in mice recovered from allergic airway inflammation.

Chronic asthma patients experience difficulties even years after the inciting allergen. Although studies in small animal asthma models have enormously advanced progress in uncovering the mechanisms of inception and development of the disease, little is known about the processes involved in the persistence of asthma symptoms in the absence of allergen exposure. Long-term asthma mouse models have so far been scarce or not been able to reproduce the findings in patients. Here we used a common ovalbumin-induced acute allergic airway inflammation mouse model to study lung function and remodeling after a 4-mo recovery period. We show by X-ray-based lung function measurements that the recovered mice continue to show impaired lung function by displaying significant air trapping compared with controls. High-resolution synchrotron phase-contrast computed tomography of structural alterations and diaphragm motion analysis suggest that these changes in pulmonary function are the result of a pronounced loss in lung elasticity. Histology of lung sections confirmed that this is most likely caused by a decrease in elastic fibers, indicating that remodeling can develop or persist independent of acute inflammation and is closely related to a loss in lung function. Our findings demonstrate that this X-ray-based imaging platform has the potential to comprehensively and noninvasively unravel long-term effects in preclinical mouse models of allergic airway inflammation and thus benefits our understanding of chronic asthma.