Nitric oxide prevents aortic valve calcification by S-nitrosylation of USP9X to activate NOTCH signaling

Area of Science:

• Biochemistry
• Molecular Biology
• Cardiovascular Research

Background:

• Calcific aortic valve disease (CAVD) is a growing health concern.
• Endothelial dysfunction contributes to CAVD pathogenesis.
• Nitric oxide (NO) was previously identified as a potential calcification inhibitor via NOTCH1 activation.

Purpose of the Study:

• To elucidate the S-nitrosylation-mediated mechanism by which NO inhibits CAVD.
• To investigate the role of USP9X in NO-dependent regulation of the NOTCH pathway in CAVD.

Main Methods:

• Porcine aortic valve interstitial cells were used to study NO's effect on calcification.
• Single-cell RNA sequencing (scRNA-seq) analyzed NO's regulation of the NOTCH pathway.
• Proteomics identified S-nitrosylated proteins, focusing on USP9X.
• Genetic deletion of Usp9x in mice and analysis of human calcified valves were performed.

Main Results:

• NO was shown to rescue calcification in valve cells through an S-nitrosylation-dependent mechanism.
• scRNA-seq confirmed NO's regulation of the NOTCH pathway.
• Proteomic analysis identified USP9X as a key S-nitrosylated protein involved in NOTCH regulation.
• S-nitrosylated USP9X deubiquitinates and stabilizes MIB1, leading to NOTCH1 activation.
• Genetic deletion of Usp9x induced CAVD in mice, and human CAVD samples showed reduced USP9X S-nitrosylation.

Conclusions:

• A novel mechanism reveals S-nitrosylation of USP9X as a critical regulator of the ubiquitin-proteasome pathway in preventing CAVD.
• This pathway involves USP9X-mediated stabilization of MIB1, promoting NOTCH1 activation and inhibiting aortic valve calcification.