Role of S-nitrosylation in regulating cardiac function and disease

National Heart, Lung, And Blood Institute

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Abstract

Modifications of protein cysteine residues have been shown to play important roles in ischemic preconditioning (IPC)-induced acute cardioprotection. During cardiac ischemia-reperfusion, excess reactive oxygen species can damage mitochondrial, cellular and organ function. We were involved in a collaborative study which showed that cysteine oxidation of the mitochondrial protein Opa1 contributes to mitochondrial damage and cell death caused by oxidative stress. Oxy-proteomics of ischemic-reperfused hearts reveal oxidation of the C-terminal C786 of Opa1 and treatment of mouse perfused hearts, adult cardiomyocytes, and fibroblasts with H2O2 leads to the formation of a reduction-sensitive 180 KDa Opa1 complex, distinct from the 270 KDa one antagonizing cristae remodeling. This Opa1 oxidation process is curtailed by mutation of C786 and of the other 3 Cys residues of its C-terminal domain (Opa1TetraCys). When reintroduced in Opa1-/- cells, Opa1TetraCys is not efficiently processed into short Opa1TetraCys and hence fails to fuse mitochondria. Unexpectedly, Opa1TetraCys restores mitochondrial ultrastructure in Opa1-/- cells and protects them from H2O2-induced mitochondrial depolarization, cristae remodeling, cytochrome c release and cell death. Thus, preventing the Opa1 oxidation occurring during heart ischemia reperfusion reduces mitochondrial damage and cell death induced by oxidative stress independently of mitochondrial fusion. We also examined whether NO signaling contributes to the sex differences observed in mitochondrial calcium accumulation. To test this hypothesis, we measured mitochondrial calcium levels in ex vivo perfused hearts from male and female mice using mt-R-GECO the previously described transmural spectroscopy method in heart perfused with modulators of NO/SNO. We found that the reduced rise in mitochondrial calcium observed in females was blocked by perfusion with an NO inhibitor and that the rise in mitochondrial calcium was enhanced in males with an NO donor. In parallel, we assessed the redox state of the mitochondrial calcium uniporter (MCU) by simultaneously measuring SNO and total protein oxidation. Our results indicate that SNO of MCU is higher in females contributing to a reduction in mitochondrial calcium accumulation, a key hallmark of ischemia/reperfusion (I/R) injury. We also developed methods to directly measure ROS using HyPer7, a genetically-encoded mitochondrial-targeted fluorescent ROS indicator, in adult mouse hearts. We used confocal microscopy to confirm that HyPer7 was expressed and localized to the mitochondrial matrix of isolated cardiomyocytes. We then validated that HyPer7 sensitively responds to mtROS by incubating isolated cardiac mitochondria with agents known to stimulate ROS production.