ABSTRACT: Pulmonary valve stenosis (PVS) is the most common congenital heart defect in Noonan syndrome (NS) and related RASopathies, yet the molecular mechanisms that link the underlying pathogenic variants to the valve pathology remains poorly defined. Here, we utilized a human iPSC-based valve differentiation platform to generate the cardiac valve cell lineage—including fibrosa and spongiosa valve interstitial cell (VIC) subtypes. Applying this model to CRISPR-edited iPSCs carrying NS- and Noonan syndrome with multiple lentigines (NSML)-associated variants, we identified early mesodermal and endocardial specification defects in all lines. Additionally, we found that NS-iPSCs exhibited fibrosa VIC-specific defects in endothelial-to-mesenchymal transition (EndMT), which was most pronounced in PTPN11N308D (N308D) cells. Through single-cell transcriptomics, we revealed widespread dysregulation of extracellular matrix (ECM) programs in N308D fibrosa VICs, including increased expression of collagens and proteoglycans, as well as dysregulation of multiple genes involved in ECM remodeling. We also detected activation of RAS-MAPK, TGFb, and fibrosis-associated pathways in our transcriptional dataset. We then characterized the global signaling changes in N308D fibrosa VICs through mass spectrometry-based phosphoproteomic profiling, which identified coordinated increases in ERK, PKC, and stress-related kinases, as well as enhanced activity at the TGFb receptor. Functionally, we observed that N308D fibrosa VICs had an exaggerated upregulation of ECM genes in the presence of TGFb2 ligand, suggesting that these cells are hypersensitive to TGFb stimulation. Furthermore, we demonstrated that this pathological ECM-program occurs independently of BAMBI, a negative regulator of TGFb signaling that was found to be decreased in N308D fibrosa VICs. Lastly, we performed histopathological analyses of stenotic pulmonary valves from two NS infants, which demonstrated marked overproduction and disorganization of ECM, mirroring the findings from our iPSC-based disease model. Together, our data reveals a central mechanism where NS-associated alleles sensitize fibrosa VICs to TGFb, which leads to aberrant downstream TGFb signaling and drives the pathological ECM program in NS-associated PVS.
