Chaperone-Mediated Autophagy of eNOS in Myocardial Ischemia-Reperfusion Injury

Rationale: Nitric oxide (NO) produced by eNOS (endothelial NO synthase) protects against myocardial ischemia-reperfusion injury (I/R). However, reperfusion of myocardium results in superoxide (O-2(center dot-)) generation, which promotes eNOS glutathionylation that produces O-2(center dot-) instead of NO. It is unclear whether SG-eNOS (glutathionylated eNOS) continues to produce O-2(center dot-) indefinitely or undergoes a time-dependent degradation. Objective: To determine whether SG-eNOS continues to produce O-2(center dot-) in I/R for a prolonged period causing accentuated I/R injury or it undergoes a time-dependent degradation. Methods and Results: Since SG-eNOS produces significant O-2(center dot-) instead of NO, we sought to determine the time-course of SG-eNOS levels in the human coronary artery endothelial cell in hypoxia/reoxygenation by Western analysis and immunoprecipitation. SG-eNOS was degraded by chaperone-mediated autophagy (CMA), as inhibitors of CMA rescued eNOS expression. We further confirmed CMA by high-resolution confocal and electron microscopy. We showed that SG-eNOS is targeted by HSC70 chaperone via its interaction with glutathionylated-cysteine 691 and 910. Glutathionylation of cysteine 691 residue in hypoxia/reoxygenation exposes 735QRYRL739 motif for interaction with HSC70, and consequent transportation to LAMP2A (lysosome-associated membrane protein 2) vesicle, where it is degraded by lysosomal proteases. Mutagenesis of these residues in eNOS inhibited its CMA. Using contrast echocardiography and electron paramagnetic resonance spectrometry, we found that Trx-Tg mice show improved myocardial perfusion and decreased myocardial apoptosis in I/R due to deglutathionylation of SG-eNOS and restoration of NO generation. Further, WT mice treated with rhTrx (recombinant human thioredoxin) were protected against eNOS CMA and restored NO production with improved myocardial perfusion and decreased I/R injury. Conclusions: SG-eNOS undergoes degradation via CMA, following prolonged retention in the cytosol. CMA of SG-eNOS terminates O-2(center dot-) generation preventing further tissue damage but causes irreversible loss of eNOS and NO availability. Prompt deglutathionylation of SG-eNOS prevents CMA, promotes NO production, and improved myocardial perfusion, resulting in amelioration of reperfusion injury.

Automated summary

In the context of myocardial ischemia-reperfusion injury, it has been discovered that SG-eNOS undergoes degradation through a specific mechanism, preventing further tissue damage but also causing irreversible loss of eNOS and NO availability.