Abstract 14610: Thoracic Aortic Aneurysm-causing Mutation, R258C, in Smooth Muscle Alpha-actin Inhibits Functional Effects of Wild-type Alpha-actin in Patient-derived Fibroblasts
Familial thoracic aortic aneurysms and dissections (TAAD) are most commonly caused by missense mutations in smooth muscle α-actin (SMαA), encoded by ACTA2. Although TAAD is hypothesized to result in part from a compromised ability of aortic smooth muscle cells (SMCs) to sense their mechanical environment, the specifics of how ACTA2 mutations affect cellular mechanotransduction remain to be defined. We established hTERT-immortalized lines of dermal fibroblasts from volunteers (WT) and patients with ACTA2 mutations (MT). SMαA expression was induced with adenovirus encoding MRTF-A, a potent co-activator of SM contractile genes, including ACTA2. We focus here on the ACTA2 R258C mutation, a recurrent mutation associated with early onset of TAAD. Quantification of actin expression by 2D-PAGE and western blotting showed that induced WT fibroblasts expressed ~40% β-, 30% γ- and 30% SM-α-actin, while heterozygous MT cells expressed ~40% β-, 30% γ-, 15% WT-SM-α- and 15% MT-SM-α-actin, with un-induced cells expressing <5% SMαA. Cytoskeletal structure was studied by immunofluorescence and confocal microscopy, and by sedimentation for actin distribution in filamentous (F) and monomeric (G) fractions. Cell motile and contractile properties were investigated by scratch-induced cell migration, trans-well migration, and collagen matrix contraction assays. As described for other models of SMαA induction in fibroblasts, we found that MRTF-A induction of SMαA in WT cells significantly increased formation of stress fibers and focal adhesions, F/G ratio (p<0.01), and matrix contraction (p<0.05), and significantly decreased cell migration in scratch-induced (p<0.01) and trans-well (p<0.0001) migration assays. In contrast, expression of MT SMαA in heterozygous R258C MT fibroblasts abrogated the effects of SMαA on formation of stress fibers and focal adhesions, F/G ratio, matrix contraction and cell migration in comparison to induction of SMαA in WT cells. These results demonstrate that R258C MT SMαA disrupts cytoskeletal functions attributed to SMαA in WT fibroblasts and are consistent with defects in mechanotransduction, which may shed light on cellular defects due to ACTA2 mutations in both aortic SMCs and adventitial fibroblasts.
Author Disclosures: Z. Liu: None. A. Chang: None. F. Grinnell: None. K. Trybus: None. D. Milewicz: None. K. Kamm: None.
- © 2015 by American Heart Association, Inc.