Abstract 16734: Thioredoxin-1 Potentiates Mtor Activity Through Direct Reduction In Cardiomyocytes
Thioredoxin 1 (Trx1) is a 12 KDa oxidoreductase, which reduces proteins with disulfide bonds through thiol-disulfide exchange reactions. Trx1 protects cardiomyocytes (CMs) against stress, such as ischemia and pressure-overload. Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, metabolism and survival. Using a substrate-trapping mutant of Trx1 (Trx1C35S), we have reported previously that mTOR may be a Trx1 substrate. However, how mTOR is regulated by the redox status and Trx1 in CMs remains to be elucidated. In the presence of hydrogen peroxide (H2O2, 100 nM), a part of mTOR exhibited a band shift to a high molecular weight in Western blot analyses using non-reducing SDS-PAGE, suggesting that mTOR is subjected to oxidative posttranslational modifications. The mTOR oxidation was accompanied by reduced phosphorylation of mTOR substrates, such as S6 kinase and 4E-BP1. Insulin-induced activation of mTOR was also inhibited in the presence of H2O2. These results suggest that the kinase activity of mTOR is inhibited by oxidation. The mTOR oxidation and inhibition by H2O2 were normalized in the presence of Trx1 overexpression, but they were enhanced by Trx1 knockdown. In order to evaluate whether Trx1 and mTOR physically interact with one another in the heart, we used cardiac-specific Trx1C35S transgenic mice. The interaction between Trx1C35S and mTOR was increased in the presence of pressure overload. H2O2 also enhanced the interaction between Trx1C35S and mTOR in cultured CMs, an effect which was diminished in the presence of dithiothreitol, a reducing agent that cleaves disulfide bonds. These results suggest that mTOR is a Trx1 substrate and that Trx1 cleaves disulfide bonds in mTOR. H2O2-induced CM death was inhibited by Trx1 overexpression, an effect which was diminished in the presence of rapamycin, an inhibitor of mTOR Complex 1 (relative cell viability: control: 1; control H2O2: 0.45; Trx1 H2O2: 1.1; Rapamycin/ H2O2: 0.52; Rapamycin/Trx1/H2O2: 0.50*, P<0.05 vs Trx1 H2O2). These results suggest that mTOR is a novel substrate of Trx1 and that Trx1 positively regulates the function of mTOR, which in turn promotes survival in CMs.
Author Disclosures: S. Oka: None. T. Hirata: None. H. Yaginuma: None. D. Shao: None. N. Nagarajan: None. J. Sadoshima: None.
- © 2016 by American Heart Association, Inc.