Abstract Background: – MYH11 encodes smooth muscle cell specific myosin heavy chain (SM-MHC), a crucial contractile protein in vascular smooth muscle cells (vSMC) responsible for force generation. Pathogenic variants in MYH11 segregate with familial thoracic aortic aneurysms and dissections (TAAD) and patent ductus arteriosus. Recently we identified a Dutch family with a MYH11 c.4599+1GA pathogenicn variant, that presented with PDA and abdominal dissections. The variant leads to disturbances in the SM-MHC protein, which affect dimerization and ultimately actin-myosin interactions. Because of this, force generation is affected, as are interactions with the extracellular matrix. This can potentially drive vSMCs into a synthetic – migratory and proliferative - phenotype aiding in disease progression.Purpose – We aim to use induced pluripotent stem cell (iPSC) model of MYH11-related familial TAAD to recapitulate the disease pathophysiology and to establish an in vitro platform for testing therapeutic strategies. We hypothesize that mutant vSMCs display a more synthetic phenotype at baseline. Methods – vSMCs were differentiated from variant carriers (n=3) and non-carrier controls (n=4) by PDGF-BB and TGF-β treatment of a neural crest precursor population. An isogenic control line was generated using CRISPR-cas9 based gene editing of a mutant iPSC line. To functionally assess the iVSMCs, we examined the expression of contractile proteins, including SM-MHC (MYH11), SM22a (TAGLN), alpha smooth muscle actin (ACTA2), calponin (CNN1) and phosphorylated myosin light chain kinase (MYLK), using immunofluorescence staining and gene expression (qPCR and ddPCR). Cell migration and stiffness were evaluated using scratch assay (Omni), transwell assay (XCelligence), and nano-indentation (Pavone), respectively. Finally, calcium handling was assessed following stimulation with the cholinergic agonist carbachol, after loading the cells with the fluorescent calcium indicator Fluo8. Results – The mutant cells showed a trend toward lower contractile protein fluorescent signal, indicating lower expression of contractile proteins in these cells. They also exhibited increased migration and significantly reduced cell stiffness, consistent with a synthetic vSMC phenotype. Calcium analysis showed larger Ca²+ peak amplitude and altered decay to baseline compared with WT cells, indicating enhanced Ca²+ release and impaired Ca²+ clearance. Conclusion – We successfully differentiated three vSMC lines from MYH11 c.4599+1GA carriers and four non-carrier controls, along with one isogenic control. The mutant vSMC lines showed a more synthetic phenotype compared with wild-type cells. These models offer an ideal platform to test gene therapies aimed at alleviating the observed phenotypic differences.
Atash et al. (Fri,) studied this question.