Abstract 393: S-nitrosylation of Atg7 by Thioredoxin-1 (trx1) Protects the Heart Against Myocardial Ischemia via Autophagy Regulation

Abstract

Thioredoxin 1 (Trx1) is an oxidoreductase that reduces proteins with disulfide bonds. Trx1 also functions as a transnitrosylase, but this occurs only when Trx1 is oxidized at Cys32 and Cys35. In cultured cardiomyocytes (CMs), glucose deprivation (GD) induces oxidation of Trx1 and Trx1 is transnitrosylated. Thus, we hypothesized that Trx1 promotes GD-induced autophagy through its function as a transnitrosylase rather than as an oxidoreductase. GD-induced autophagy, evaluated by counting GFP-LC3 puncta, was inhibited in the presence of the transnitrosylation-defective Trx1C73S mutant (GFP-LC3 puncta per cell under GD; control: 38; Trx1 knockdown: 2*; Trx1C73S: 17*; p<0.05 vs. control), suggesting that Cys73 in Trx1 plays an important role in mediating GD-induced autophagy. Mass spectrometric analyses and biochemical assays showed that Atg7, an essential autophagy regulator, is a Trx1 target and that Trx1 binds to Atg7 via Cys454 and Cys458 in Atg7, thereby transnitrosylating Atg7 at Cys294 and Cys402. Trx1C73S knock-in (Trx1C73S-KI) promoted coronary ligation (CL)-induced myocardial infarction (MI) (MI size/area at risk (AAR) (%); Wild type (WT): 21; Trx1C73S-KI: 42*; p<0.05 vs. WT), in association with reduced S-nitrosylation of Atg7. To test the role of S-nitrosylation of Atg7 in mediating autophagy, we transduced an S-nitrosylation defective Atg7 mutant (Atg7CC294/402SS) into adult cardiomyocytes isolated from cardiac-specific Atg7 knockout mice. Compared to intact Atg7, Atg7CC294/402SS was less able to induce autophagy, as evidenced by reduced LC-3II formation (relative LC3-II; intact Atg7: 1.0; Atg7CC294/402SS: 0.81*; p<0.05). Atg7C402S, but not Atg7C294S, knock-in exacerbated CL-induced MI (MI size/AAR (%); WT: 32; Atg7C402-KI: 42*; p<0.05 vs. WT). These results suggest that Trx1 protects the heart against MI by mediating autophagy via S-nitrosylation of Atg7 at Cys402.