Abstract 19429: Trx1 Regulates Nitroso-redox Balance to Stimulate Autophagy and Cell Survival in Cardiomyocytes

Abstract

Thioredoxin-1 (Trx1) is cardioprotective during oxidative stress. In addition to acting as an oxidoreductase, Trx1 also trans-nitrosylates other proteins. However, the role of Trx1-dependent S-nitrosylation in cardiomyocytes (CMs) is unknown. We investigated how Trx1 affects protein S-nitrosylation (SNO) and the nitroso-redox balance to regulate CM survival during glucose deprivation (GD). Mass spectrometry (MS/MS) analysis showed that Trx1 is S-nitrosylated at Cys73. Protein SNO levels were increased after 4 hrs of GD (1.64 fold, p<0.05; biotin switch assay). Overexpression of Trx1WT increased (3.94 fold), whereas knockdown of Trx1 (0.66 fold, p<0.01) or overexpression of Trx1C73S (0.77 fold, p<0.01) decreased, total protein SNO in response to GD, suggesting that Trx1 Cys73 regulates protein SNO in CMs during GD. Overexpression of Trx1 increased CM survival 24 hrs after GD (1.42 fold vs LacZ, p<0.05). Conversely, shTrx1 (2.13 fold vs control, p<0.01) or Trx1C73S (1.73 fold vs LacZ, p<0.01) increased cell death during GD. Either knockdown of Trx1 (LC3-II/Tubulin, 0.55 fold vs control) or overexpression of Trx1C73S (vs LacZ: LC3-II/Tubulin, 0.60 fold; autophagosomes, 0.83 fold, p<0.005; autolysosomes, 0.62 fold, p<0.005) significantly decreased autophagosome formation during GD. Increased interaction between Trx1 and a key autophagy protein, Atg7, was observed during GD and oxidative stress, as evaluated with the Trx1C35S trapping mutant. However, addition of DTT abolished this interaction, suggesting that Trx1 and Atg7 interact with one another via a disulfide linkage. By biotin switch assay and MS/MS analyses, we found that SNO-Trx1 can trans-nitrosylate Atg7 in vitro and that Atg7 is S-nitrosylated during GD in CMs. By labeling reduced and oxidized Cys residues in Atg7 with iodoacetamide and MMTS respectively, we determined that the level of Atg7 oxidation did not increase during oxidative stress in CMs, suggesting that Trx1 trans-nitrosylates Atg7 to prevent irreversible oxidative damage in Atg7. Taken all together, our results indicate that Trx1 promotes trans-nitrosylation of cellular proteins such as Atg7 to regulate the nitroso-redox balance and thus promote cell survival by stimulating autophagy during energy stress in CMs.