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.

Birth defects are the leading cause of infant mortality, and they are caused by a combination of genetic and environmental factors. Environmental risk factors may contribute to birth defects in genetically susceptible infants by altering critical molecular pathways during embryogenesis, but experimental evidence for gene-environment interactions is limited. Fetal hyperglycemia associated with maternal diabetes results in a 5-fold increased risk of congenital heart disease (CHD), but the molecular basis for this correlation is unknown. Here, we show that the effects of maternal hyperglycemia on cardiac development are sensitized by haploinsufficiency of Notch1 , a key transcriptional regulator known to cause CHD. Using ATAC-seq, we found that hyperglycemia decreased chromatin accessibility at the endothelial NO synthase ( Nos3 ) locus, resulting in reduced NO synthesis. Transcription of Jarid2, a regulator of histone methyltransferase complexes, was increased in response to reduced NO, and this upregulation directly resulted in inhibition of Notch1 expression to levels below a threshold necessary for normal heart development. We extended these findings using a Drosophila maternal diabetic model that revealed the evolutionary conservation of this interaction and the Jarid2-mediated mechanism. These findings identify a gene-environment interaction between maternal hyperglycemia and Notch signaling and support a model in which environmental factors cause birth defects in genetically susceptible infants.

Congenital heart disease (CHD) is the most prevalent structural malformations of the heart affecting ∼1% of live births. To date, both damaging genetic variations and adverse environmental exposure such as maternal diabetes have been found to cause CHD. Clinical studies show ∼fivefold higher risk of CHD in the offspring of mothers with pregestational diabetes. Maternal pregestational diabetes affects the gene regulatory networks key to proper cardiac development in the fetus. However, the cell-type specificity of these gene regulatory responses to maternal diabetes and their association with the observed cardiac defects in the fetuses remains unknown. To uncover the transcriptional responses to maternal diabetes in the early embryonic heart, we used an established murine model of pregestational diabetes. In this model, we have previously demonstrated an increased incidence of CHD. Here, we show maternal hyperglycemia (matHG) elicits diverse cellular responses during heart development by single-cell RNA-sequencing in embryonic hearts exposed to control and matHG environment. Through differential gene-expression and pseudotime trajectory analyses of this data, we identified changes in lineage specifying transcription factors, predominantly affecting Isl1 ⁺ second heart field progenitors and Tnnt2 ⁺ cardiomyocytes with matHG. Using in vivo cell-lineage tracing studies, we confirmed that matHG exposure leads to impaired second heart field-derived cardiomyocyte differentiation. Finally, this work identifies matHG-mediated transcriptional determinants in cardiac cell lineages elevate CHD risk and show perturbations in Isl1 -dependent gene-regulatory network ( Isl1-GRN ) affect cardiomyocyte differentiation. Functional analysis of this GRN in cardiac progenitor cells will provide further mechanistic insights into matHG-induced severity of CHD associated with diabetic pregnancies.

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 that NO rescues calcification by a S-nitrosylation-mediated mechanism in porcine aortic valve interstitial cells (pAVICs) and single cell RNA-seq demonstrated regulation of NOTCH pathway by NO. A unbiased proteomic approach to identify S-nitrosylated proteins in valve cells found enrichment of the ubiquitin proteasome pathway and implicated S-nitrosylation of USP9X 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 aortic valve disease and human calcified aortic valves displayed reduced S-nitrosylation of USP9X. These results demonstrate a novel mechanism by which S-nitrosylation dependent regulation of ubiquitin-associated pathway prevents CAVD.

BK _Ca channels are large-conductance calcium and voltage-activated potassium channels that are heterogeneously expressed in a wide array of cells. Activation of BK _Ca channels present in mitochondria of adult ventricular cardiomyocytes is implicated in cardioprotection against ischemia-reperfusion (IR) injury. However, the BK _Ca channel’s activity has never been detected in the plasma membrane of adult ventricular cardiomyocytes. In this study, we report the presence of the BK _Ca channel in the plasma membrane and mitochondria of neonatal murine and rodent cardiomyocytes which protects the heart on inhibition but not activation. Furthermore, K ⁺ currents measured in neonatal cardiomyocyte (NCM) was sensitive to iberiotoxin (IbTx), suggesting the presence of BK _Ca channels in the plasma membrane. Neonatal hearts subjected to IR when post-conditioned with NS1619 during reoxygenation increased the myocardial infarction whereas IbTx reduced the infarct size. In agreement, isolated NCM also presented increased apoptosis on treatment with NS1619 during hypoxia and reoxygenation, whereas IbTx reduced TUNEL positive cells. In NCMs, activation of BK _Ca channels increased the intracellular reactive oxygen species post HR injury. Electrophysiological characterization of NCMs indicated that NS1619 increased the beat period, field, and action potential duration, and decreased the conduction velocity and spike amplitude. In contrast, IbTx had no impact on the electrophysiological properties of NCMs. Taken together, our data established that inhibition of plasma membrane BK _Ca channels in the NCM protects neonatal heart/cardiomyocytes from IR injury. Furthermore, the functional disparity observed towards the cardioprotective activity of BK _Ca channels in adults compared to neonatal heart could be attributed to their differential localization.