Abstract 150: S-nitrosylation Of Thioredoxin1 At Cysteine 73 Promotes Trans-nitrosylation, Autophagy And Cell Survival During Glucose Deprivation.
Thioredoxin-1 (Trx1) is a key antioxidant protein that is known to play a protective role in the heart during oxidative stress mainly through its oxidoreductase activity. Trx1 can be S-nitrosylated and, in turn, can trans-nitrosylate other proteins. However, the role of Trx1-dependent S-nitrosylation in cardiomyocytes (CMs) is not known. Here, we investigated the role of Trx1-mediated protein S-nitrosylation in the regulation of CM survival in response to stress. Using the biotin-switch assay, we found that wild-type Trx1 (Trx1WT) was S-nitrosylated, whereas the extent of S-nitrosylation was attenuated in Trx1C73S, suggesting that Trx1 is S-nitrosylated at Cys73. Also, we observed that the redox activity of Trx1 was intact in the Trx1C73S mutant. Overall protein S-nitrosylation in rat neonatal CMs was increased in response to 4 hrs of glucose deprivation (GD). Using biotin-switch assay and immunocytochemistry (fluorescent staining of s-nitrosylated cysteines), we observed that overexpression of Trx1WT increased, whereas short-hairpin RNA-mediated knockdown of Trx1 (shTrx1) or overexpression of Trx1C73S decreased, total protein S-nitrosylation in response to GD. These results suggest that Trx1Cys73 plays a key role in the regulation of protein S-nitrosylation in CMs during GD. Overexpression of Trx1 increased CM survival after 24 hrs of GD (Trx1WT vs. LacZ: propidium iodide assay, 0.5 ± 0.08-fold, p<0.01). Conversely, shTrx1 or overexpression of Trx1C73S increased cell death during GD (Trx1C73S vs. LacZ: 1.7 ± 0.034-fold, p<0.05). Autophagy is a pro-survival mechanism during GD. Therefore, we tested the effect of Trx1 on autophagy. After 4 hrs of GD, knockdown of Trx1 or overexpression of Trx1C73S decreased autophagy compared to control cells (LC3-II/LC3-I, 0.7-fold; autophagosomes, 0.83 ± 0.16-fold; autolysosomes, 0.62 ± 0.13-fold, p<0.005). Taken altogether, our results suggest that Trx1 promotes autophagy during GD through a trans-nitrosylation dependent mechanism. S-nitrosylation of Trx1 at Cys73 is associated with an overall increase in protein S-nitrosylation in CMs and promotes autophagy and thus, cell survival during GD.
Author Disclosures: N. Nagarajan: None S. Sciarretta: None J. Sadoshima: None.
- © 2014 by American Heart Association, Inc.