No abstract

Calcific aortic valve disease (CAVD) is an increasingly prevalent condition, and endothelial dysfunction is implicated in its etiology. We previously identified nitric oxide (NO) as a calcification inhibitor by its activation of NOTCH1, which is genetically linked to human CAVD. Here, we show NO rescues calcification by an S-nitrosylation–mediated mechanism in porcine aortic valve interstitial cells and single-cell RNA-seq demonstrated NO regulates the NOTCH pathway. An unbiased proteomic approach to identify S-nitrosylated proteins in valve cells found enrichment of the ubiquitin-proteasome pathway and implicated S-nitrosylation of USP9X (ubiquitin specific peptidase 9, X-linked) in NOTCH regulation during calcification. Furthermore, S-nitrosylated USP9X was shown to deubiquitinate and stabilize MIB1 for NOTCH1 activation. Consistent with this, genetic deletion of Usp9x in mice demonstrated CAVD and human calcified aortic valves displayed reduced S-nitrosylation of USP9X. These results demonstrate a previously unidentified mechanism by which S-nitrosylation–dependent regulation of a ubiquitin-associated pathway prevents CAVD.

Current models entail that transport through the Golgi — the main sorting compartment of the cell — occurs via cisternal progression/maturation, and that Ypt/Rab GTPases regulate this process. However, there is very limited evidence that cisternal progression is regulated, and no evidence for involvement of Ypt/Rab GTPases in such a regulation. Moreover, controversy about the placement of two of the founding members of the Ypt/Rab family, Ypt1 and Ypt31, to specific Golgi cisternae interferes with addressing this question in yeast, where cisternal progression has been extensively studied. Here, we establish the localization of Ypt1 and Ypt31 to opposite faces of the Golgi, early and late, respectively. Moreover, we show that they partially overlap on a transitional compartment. Finally, we determine that changes in Ypt1 and Ypt31 activity affect Golgi cisternal progression, early-to- transitional and transitional-to-late, respectively. These results show that Ypt/Rab GTPases regulate two separate steps of Golgi cisternal progression.

BackgroundCongenital heart disease is the most common type of birth defect, affecting ≈2% of the population. Malformations involving the cardiac outflow tract and semilunar valves account for >50% of these cases predominantly because of a bicuspid aortic valve, which has an estimated prevalence of 1% in the population. We previously reported that mutations in NOTCH1 were a cause of bicuspid aortic valve in nonsyndromic autosomal‐dominant human pedigrees. Subsequently, we described a highly penetrant mouse model of aortic valve disease, consisting of a bicuspid aortic valve with thickened cusps and associated stenosis and regurgitation, in Notch1‐haploinsufficient adult mice backcrossed into a Nos3‐null background.Methods and ResultsHere, we described the congenital cardiac abnormalities in Notch1 +/− ;Nos3 −/− embryos that led to ≈65% lethality by postnatal day 10. Although expected Mendelian ratios of Notch1 +/− ;Nos3 −/− embryos were found at embryonic day 18.5, histological examination revealed thickened, malformed semilunar valve leaflets accompanied by additional anomalies of the cardiac outflow tract including ventricular septal defects and overriding aorta. The aortic valve leaflets of Notch1 +/− ;Nos3 −/− embryos at embryonic day 15.5 were significantly thicker than controls, consistent with a defect in remodeling of the semilunar valve cushions. In addition, we generated mice haploinsufficient for Notch1 specifically in endothelial and endothelial‐derived cells in a Nos3‐null background and found that Notch1 fl/+;Tie2‐Cre +/− ;Nos3 −/− mice recapitulate the congenital cardiac phenotype of Notch1 +/− ;Nos3 −/− embryos.ConclusionsOur data demonstrate the role of endothelial Notch1 in the proper development of the semilunar valves and cardiac outflow tract.