RESUMEN
Redox signaling and cardiac function are tightly linked. However, it is largely unknown which protein targets are affected by hydrogen peroxide (H2O2) in cardiomyocytes that underly impaired inotropic effects during oxidative stress. Here, we combine a chemogenetic mouse model (HyPer-DAO mice) and a redox-proteomics approach to identify redox sensitive proteins. Using the HyPer-DAO mice, we demonstrate that increased endogenous production of H2O2 in cardiomyocytes leads to a reversible impairment of cardiac contractility in vivo. Notably, we identify the γ-subunit of the TCA cycle enzyme isocitrate dehydrogenase (IDH)3 as a redox switch, linking its modification to altered mitochondrial metabolism. Using microsecond molecular dynamics simulations and experiments using cysteine-gene-edited cells reveal that IDH3γ Cys148 and 284 are critically involved in the H2O2-dependent regulation of IDH3 activity. Our findings provide an unexpected mechanism by which mitochondrial metabolism can be modulated through redox signaling processes.
Asunto(s)
Peróxido de Hidrógeno , Mitocondrias , Ratones , Animales , Peróxido de Hidrógeno/metabolismo , Mitocondrias/metabolismo , Oxidación-Reducción , Metabolismo Energético , Miocitos Cardíacos/metabolismo , Estrés OxidativoAsunto(s)
Infarto del Miocardio/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Interferencia de ARN , Animales , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Infarto del Miocardio/genética , Infarto del Miocardio/terapia , Tratamiento con ARN de Interferencia/métodosRESUMEN
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¯0.4â¯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¯8.7â¯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.