Nuclear Smooth Muscle a-actin Is Critical For Smooth Muscle Cell Differentiation And To Prevent Cerebrovascular Disease

Missense pathogenic variants in ACTA2, encoding α-smooth muscle actin (SMA), predispose to thoracic aortic aneurysms. A subset of these ACTA2 pathogenic variants also predispose to childhood onset cerebrovascular disease characterized by occlusion of the distal internal carotid arteries and small vessel disease. In our studies to identify how specific ACTA2 pathogenic variants predispose to cerebrovascular disease, we confirmed that SMA is localized to the nucleus in wildtype (WT) smooth muscle cells (SMCs), is enriched in the nucleus over β-actin with differentiation of SMCs, associates with the INO80 chromatin remodeling complex, and selectively binds to the promoters of SMC contractile genes. Pluripotent stem cell-derived SMCs from patients with ACTA2 -associated cerebrovascular disease ( ACTA2 R179C) as well as SMCs explanted from a knockin mouse model ( Acta2 SMC-R179C/+ ) confirm that R179 variants disrupt SMA nuclear localization. Both mouse and human cells harboring the R179C pathogenic variant proliferate and migrate more than WT SMCs, are less differentiated than WT SMCs, and have increased expression of pluripotency-associated genes. Although the variant is heterozygous, these cells show a dominant negative impact on nuclear SMA function through dramatically reduced levels of SMA in the nucleus, in the INO80 chromatin remodeling complex, and on the promoters of SMC-specific genes. Finally, single cell RNA sequencing analysis of aortic tissue from an ACTA2 R179H patient confirms dedifferentiation of SMCs as a key phenotype associated with this mutation. Taken together, we have identified a novel role for α-SMA in driving differentiation of SMCs, and our data supports that defects in this nuclear role drive cellular phenotypes consistent with the cerebrovascular disease seen in patients with ACTA2 R179 pathogenic variants.