Dysregulated TGFβ-ERK Signaling Drives Aberrant Extracellular Matrix Production in Noonan Syndrome-Associated Pulmonary Valve Stenosis

Pulmonary valve stenosis (PVS) is the most common congenital heart defect in Noonan syndrome (NS) and related RASopathies, yet the molecular mechanisms linking pathogenic variants to the valve pathology remain poorly defined. Here, we utilized a human iPSC-based valve differentiation platform to generate the cardiac valve cell lineages-including fibrosa and spongiosa valve interstitial cell (VIC) subtypes. CRISPR-edited iPSCs harboring NS gain-of-function RAS/MAPK and Noonan syndrome with multiple lentigines (NSML) dominant-negative RAS/MAPK variants exhibited early defects in mesodermal and endocardial specification in all genotypes. Additionally, NS-iPSC endocardial cells exhibited defects in endothelial-to-mesenchymal transition (EndMT) specifically towards fibrosa VICs, which was most pronounced in <i>PTPN11</i> ^<i>N308D</i> (N308D) cells. Single-cell transcriptomics 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, TGFβ, and fibrosis-associated pathways in our transcriptional dataset. Mass spectrometry-based phosphoproteomics confirmed coordinated increases in ERK, PKC, and stress-related kinases, as well as enhanced activity of the TGFβ receptor. Functionally, N308D fibrosa VICs exhibited exaggerated upregulation of ECM genes in the presence of TGFβ2 ligand, suggesting that these cells are hypersensitive to TGFβ stimulation. Furthermore, we demonstrated that this pathological ECM-program occurs independently of <i>BAMBI</i>, a negative regulator of TGFβ 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 reveal a central mechanism where NS-associated alleles sensitize fibrosa VICs to TGFβ, which leads to aberrant downstream signaling and drives the pathological ECM program in NS-associated PVS.