Abstract 20399: Trx1 Regulates Nitroso-Redox Balance to Regulate Cell Survival in Cardiomyocytes

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Background: Thioredoxin1 (Trx1) is cardioprotective during ischemia by reducing oxidized proteins through thiol-disulfide exchange reactions. Whether Trx1 also regulates nitrosylation of target proteins is not well understood in cardiomyocytes (CMs).

Goals: We investigated whether Trx1 acts a transnitrosylase and modulates the function of cardiomyocytes.

Results: Trx1 can be S-nitrosylated at Cys73 and, in turn, function as a trans-nitrosylase to transfer the -NO group to target proteins in HeLa cells. Overexpression of Trx1C73S in CMs resulted in increased cell death during 24hrs glucose deprivation (GD), as evaluated by propidium iodide staining (1.73-fold vs. LacZ-GD, p<0.01). Transgenic mice with cardiac-specific expression of Trx1(C73S) (Trx1 C73S-Tg) subjected to 3hrs of ischemia showed an increased infarct area compared to non-transgenic mice (1.5-fold, p<0.05). Taken altogether, Trx1 Cys73 plays an essential role in mediating the cardioprotective effect of Trx1. Overexpression of Trx1 C73S inhibited autophagy during GD in CMs, similar to knockdown of Trx1 (vs. LacZ: LC3-II/Tubulin: 0.60-fold; autophagosomes: 0.83-fold, p<0.005; autolysosomes: 0.62-fold, p<0.005). Trx1 interacts with Atg7, a key regulator of autophagy. Although this interaction observed in the Trx1C35S mutant, a Trx1 substrate trapping mutant, was enhanced during oxidative stress, it was abolished in the presence of DTT, a reducing agent that removes disulfide bonds. We were also able to observe enhanced Trx1-Atg7 interaction in Trx1 C35S-Tg mice subjected to ischemia. These results suggest that Trx1 and Atg7 interact via a disulfide linkage. Interestingly, Atg7 was S-nitrosylated after 30 mins of ischemia in WT mice. Similarly, S-nitrosylation of Atg7 was observed during 4hrs of GD in CMs. Using recombinant proteins, we were able to show that Trx1 trans-nitrosylates Atg7 in vitro. Furthermore, the presence of Trx1 Cys73 was essential for S-nitrosylation of Atg7 in CMs.

Conclusions: Taken altogether, our results indicate that Trx1 via Cys73 promotes trans-nitrosylation of cellular proteins, including Atg7, in order to regulate the nitroso-redox balance, thereby promoting cell survival during energy stress in CMs.

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