ABSTRACT Supravalvular aortic stenosis (SVAS) is characterized by focal narrowing of the aorta that increases the risk for sudden cardiac death. SVAS is caused by mutations in the elastin gene that lead to decreased elastin amounts and there are currently no pharmaceutical treatments. The mechanisms by which elastin insufficiency cause SVAS are not well understood. Elastin is a critical mechanical component of the aorta and contributes to the passive stiffness (or modulus) that determines how much the aorta will deform (or strain) under applied hemodynamic stresses. Strain on smooth muscle cells (SMCs) within the aortic wall affects differentiation, proliferation, and migration. Cellular transmembrane channels, including Piezo1/2, are mechanosensitive molecules that transduce mechanical changes (such as strain) into biological effects (such as differentiation). Activation of Piezo channels leads to increases in intracellular calcium that can stimulate nuclear translocation of YAP/TAZ, which are transcriptional regulators of target genes including Ctgf. Ctgf is a known modulator of SMC phenotype that encourages dedifferentiation, migration, and proliferation - all characteristics affected by strain that may contribute to SVAS. Preliminary data in our unique SVAS mouse model (TaglnCre;Elnf/f) show a reduced aortic modulus that may increase SMC strain, increased Piezo2 and Ctgf expression in aortic SMCs, and a dedifferentiated aortic SMC phenotype. We hypothesize that SVAS is caused by altered SMC mechanotransduction when enough elastin is not laid down to stiffen the aortic wall and prevent increased SMC strain as stress increases with blood pressure during development. Increased SMC strain causes overexpression/activation of Piezo2, leading to increased intracellular calcium, nuclear translocation of YAP/TAZ, and increased Ctgf transcription that causes SMC phenotype modulation contributing to stenosis. We will address our hypothesis through three complementary aims using TaglnCre;Elnf/f mice and human SMCs derived from induced pluripotent stem cells from SVAS patients. In Aim 1, we will measure the global and local elastic modulus of TaglnCre;Elnf/f aorta and SMC strain under physiologic loading conditions at different developmental time points (before and after stenosis formation) and correlate these results with changes in SMC phenotype as measured by single cell RNA-Seq. In Aim 2, we will apply strain to mouse aorta and mouse and human SMCs and measure Piezo2 expression and activity. We will chemically and genetically alter Piezo2 expression/activity and determine effects on in vitro calcium signaling and in vivo stenosis severity. In Aim 3, we will chemically and genetically manipulate Piezo2 expression/activity, YAP/TAZ localization, and Ctgf amounts in mouse aorta and mouse and human SMCs and determine the effects on SMC phenotype and stenosis severity. Our results will be important for identifying new pharmaceutical strategies that may pr...