Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia
Narayani Nagarajan, Shin-ichi Oka, Jihoon Nah, Changgong Wu, Peiyong Zhai, Risa Mukai, Xiaoyong Xu, Sanchita Kashyap, Chun-Yang Huang, Eun-Ah Sung, Wataru Mizushima, Allen Sam Titus, Koichiro Takayama, Youssef Mourad, Jamie Francisco, Tong Liu, Tong Chen, Hong Li, Junichi Sadoshima
Narayani Nagarajan, Shin-ichi Oka, Jihoon Nah, Changgong Wu, Peiyong Zhai, Risa Mukai, Xiaoyong Xu, Sanchita Kashyap, Chun-Yang Huang, Eun-Ah Sung, Wataru Mizushima, Allen Sam Titus, Koichiro Takayama, Youssef Mourad, Jamie Francisco, Tong Liu, Tong Chen, Hong Li, Junichi Sadoshima
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Research Article Cardiology Cell biology

Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia

Abstract

Modification of cysteine residues by oxidative and nitrosative stress affects structure and function of proteins, thereby contributing to the pathogenesis of cardiovascular disease. Although the major function of thioredoxin 1 (Trx1) is to reduce disulfide bonds, it can also act as either a denitrosylase or transnitrosylase in a context-dependent manner. Here we show that Trx1 transnitrosylates Atg7, an E1-like enzyme, thereby stimulating autophagy. During ischemia, Trx1 was oxidized at Cys32-Cys35 of the oxidoreductase catalytic center and S-nitrosylated at Cys73. Unexpectedly, Atg7 Cys545-Cys548 reduced the disulfide bond in Trx1 at Cys32-Cys35 through thiol-disulfide exchange and this then allowed NO to be released from Cys73 in Trx1 and transferred to Atg7 at Cys402. Experiments conducted with Atg7 C402S–knockin mice showed that S-nitrosylation of Atg7 at Cys402 promotes autophagy by stimulating E1-like activity, thereby protecting the heart against ischemia. These results suggest that the thiol-disulfide exchange and the NO transfer are functionally coupled, allowing oxidized Trx1 to mediate a salutary effect during myocardial ischemia through transnitrosylation of Atg7 and stimulation of autophagy.

Authors

Narayani Nagarajan, Shin-ichi Oka, Jihoon Nah, Changgong Wu, Peiyong Zhai, Risa Mukai, Xiaoyong Xu, Sanchita Kashyap, Chun-Yang Huang, Eun-Ah Sung, Wataru Mizushima, Allen Sam Titus, Koichiro Takayama, Youssef Mourad, Jamie Francisco, Tong Liu, Tong Chen, Hong Li, Junichi Sadoshima

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Figure 2

Trx1 Cys73 promotes autophagy during glucose deprivation.

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Trx1 Cys73 promotes autophagy during glucose deprivation. (A) Cardiomyoc...
(A) Cardiomyocytes were transduced with the indicated adenovirus along with Ad-mRFP-GFP-LC3 for 48 hours. Cardiomyocytes were cultured in normal or glucose-free medium for 4 hours. Autophagosomes and autolysosomes (yellow and free red puncta) were counted and quantified. n = 3. (B) Autophagic flux was assessed using Ad-GFP-LC3-RFP. Indicated adenovirus vectors were transduced into cardiomyocytes. The cells were incubated with glucose-free medium for 4 hours. The GFP/RFP ratio is shown. n = 3. Scale bar: 20 μm. (C) Trx1-C73S–KI mice exhibited normal cardiac function under basal and starvation conditions. n = 8. (D) Trx1 Cys73 mediates starvation-induced autophagy in the heart. LC3-II formation was evaluated after 48 hours of starvation with chloroquine (CQ) treatment. n = 6–8. (E) Trx1-C73S–KI mice and WT mice were subjected to ischemia for 3 hours. Representative images of TTC staining are shown. Scale bar: 5 mm. The percentage infarct area/area at risk (AAR) and AAR (%) are shown. (F) Echocardiographic measurements after 3 hours of ischemia. n = 5–6. (G) Plasma troponin T levels were evaluated after 3 hours of ischemia. n = 4–5. Error bars represent SEM. *P < 0.05; **P < 0.01 by 1-way ANOVA (A–D and F) or 2-tailed Student’s t test (E and G).