Thioredoxin Uses a GSH-independent Route to Deglutathionylate Endothelial Nitric-oxide Synthase and Protect against Myocardial Infarction

Abstract

Reversible glutathionylation plays a critical role in protecting protein function under conditions of oxidative stress generally and for endothelial nitric-oxide synthase (eNOS) specifically. Glutathione-dependent glutaredoxin-mediated deglutathionylation of eNOS has been shown to confer protection in a model of heart damage termed ischemia-reperfusion injury, motivating further study of eNOS deglutathionylation in general. In this report, we present evidence for an alternative mechanism of deglutathionylation. In this pathway thioredoxin (Trx), a small cellular redox protein, is shown to rescue eNOS from glutathionylation during ischemia-reperfusion in a GSH-independent manner. By comparing mice with global overexpression of Trx and mice with cardiomyocyte-specific overexpression of Trx, we demonstrate that vascular Trx-mediated deglutathionylation of eNOS protects against ischemia-reperfusion-mediated myocardial infarction. Trx deficiency in endothelial cells promoted eNOS glutathionylation and reduced its enzymatic activity, whereas increased levels of Trx led to deglutathionylated eNOS. Thioredoxin-mediated deglutathionylation of eNOS in the coronary artery in vivo protected against reperfusion injury, even in the presence of normal levels of GSH. We further show that Trx directly interacts with eNOS, and we confirmed that Cys-691 and Cys-910 are the glutathionylated sites, as mutation of these cysteines partially rescued the decrease in eNOS activity, whereas mutation of a distal site, Cys-384, did not. Collectively, this study shows for the first time that Trx is a potent deglutathionylating protein in vivo and in vitro that can deglutathionylate proteins in the presence of high levels of GSSG in conditions of oxidative stress.

Rapid free thiol rebound is a physiological response following cold‐induced vasoconstriction in healthy humans, primary Raynaud and systemic sclerosis

“…In a previously conducted experimental study in mice, the protective mechanism in endothelial function following I / R of thioredoxin, a disulfide‐reducing protein, was shown (Subramani et al. ). A similar mechanism could also play a role in hypothermia induced Raynaud's attack.…”

Thioredoxin reverses age-related hypertension by chronically improving vascular redox and restoring eNOS function

“…Thus, high amounts of TRX not only prevent eNOS glutathionylation but also continually deglutathionylate eNOS during the aging process, resulting in proper function of eNOS and production of NO rather than O 2 •− . TRX is an efficient deglutathionylating agent even in the presence of high amounts of GSSG in a myocardial ischemia–reperfusion injury model (43). In addition, we observed a consistent decrease in the amount of NOX4 in dnTrx-Tg mice but not in Trx-Tg mice.…”

Endothelial cell–cardiomyocyte crosstalk in heart development and disease

“…Generation of peroxynitrite is maximal in conditions of oxidative stress, when high levels of superoxide anion react with NO. At the same time, ECs are able to protect CM from oxidative stress by modulating several buffering systems at different levels, such as ATP-binding cassette transporter subfamily G member 2 (ABCG2) for the secretion of glutathione (Higashikuni et al 2012), NRG-1 (Kuramochi et al 2004) (see also below), tetrahydrobiopterin (BH4) (Leucker et al 2013) and thioredoxin (Trx) (Subramani et al 2016). While most of these buffering systems have been discovered in conditions of cardiac stress (i.e.…”