Redox modification of CLOCK mediates the regulatory function of endogenous H2O2 in mouse cellular respiration

Objective To explore the mechanisms of transcription factor circadian locomalor output cycles kaput(CLOCK) in mediating regulatory function of endogenous hydrogen peroxide(H2O2) for cell respiration. Methods Cellular oxygen consumption capacity and glycolysis in ClockC195S mouse embryonic fibroblasts (MEFs) and adult fibroblasts(MAFs) were tested by Agilent Seahorse XF Cell Mito Stress Test Kit and Glycolysis Stress Test Kit. The expression of key genes participating in cell respiration was detected by q-PCR. The endogenous H2O2 levels and the redox modification of CLOCK were detected by Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit and biotin-conjugated iodoacetamide (BIAM) were used respectively after the treatment of antioxidant Trolox. Changes in the oxygen consumption capacity and in key respiratory gene expression after the treatment of antioxidant Trolox were also tested in Clockwt and ClockC195S MEFs. Results Cellular oxygen consumption capacity, glycolysis and the expression of NMNAT2, a key enzyme in nicotinamide adenine denudeotide(NAD) biosynthesis, as well as NAD content all decreased in ClockC195S MEFs. The treatment with Trolox decreased endogenous H2O2 level and dampened respiration capacity in Clockwt MEFs but not in ClockC195S MEFs. Conclusions CLOCK mediates the regulation of endogenous H2O2 for cellular respiration through its redox modification at Cys195

Oxidative stress in atrial fibrillation: an emerging role of NADPH oxidase.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Patients with AF have up to seven-fold higher risk of suffering from ischemic stroke. Better understanding of etiologies of AF and its thromboembolic complications are required for improved patient care, as current anti-arrhythmic therapies have limited efficacy and off target effects. Accumulating evidence has implicated a potential role of oxidative stress in the pathogenesis of AF. Excessive production of reactive oxygen species (ROS) is likely involved in the structural and electrical remodeling of the heart, contributing to fibrosis and thrombosis. In particular, NADPH oxidase (NOX) has emerged as a potential enzymatic source for ROS production in AF based on growing evidence from clinical and animal studies. Indeed, NOX can be activated by known upstream triggers of AF such as angiotensin II and atrial stretch. In addition, treatments such as statins, antioxidants, ACEI or AT1RB have been shown to prevent post-operative AF; among which ACEI/AT1RB and statins can attenuate NOX activity. On the other hand, detailed molecular mechanisms by which specific NOX isoform(s) are involved in the pathogenesis of AF and the extent to which activation of NOX plays a causal role in AF development remains to be determined. The current review discusses causes and consequences of oxidative stress in AF with a special focus on the emerging role of NOX pathways