Loss of smooth muscle α-actin effects on mechanosensing and cell–matrix adhesions

Mutations in ACTA2, encoding smooth muscle α-actin, are a frequent cause of heritable thoracic aortic aneurysm and dissections. These mutations are associated with impaired vascular smooth muscle cell function, which leads to decreased ability of the cell to sense matrix-mediated mechanical stimuli....

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Published in:	Experimental biology and medicine (Maywood, N.J.) Vol. 245; no. 4; pp. 374 - 384
Main Authors:	Massett, MP, Bywaters, BC, Gibbs, HC, Trzeciakowski, JP, Padgham, S, Chen, J, Rivera, G, Yeh, AT, Milewicz, DM, Trache, A
Format:	Journal Article
Language:	English
Published:	London, England SAGE Publications 01-02-2020
Subjects:	Actins - deficiency Actins - metabolism Animals Aorta - metabolism Cell-Matrix Junctions - metabolism Collagen - metabolism Extracellular Matrix - metabolism Imaging, Three-Dimensional Mechanotransduction, Cellular Mice, Inbred C57BL Microscopy, Fluorescence Muscle Relaxation Muscle, Smooth - metabolism Original Research Acta2−/− mice actin vascular smooth muscle cells Integrins
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Summary:	Mutations in ACTA2, encoding smooth muscle α-actin, are a frequent cause of heritable thoracic aortic aneurysm and dissections. These mutations are associated with impaired vascular smooth muscle cell function, which leads to decreased ability of the cell to sense matrix-mediated mechanical stimuli. This study investigates how loss of smooth muscle α-actin affects cytoskeletal tension development and cell adhesion using smooth muscle cells explanted from aorta of mice lacking smooth muscle α-actin. We tested the hypothesis that reduced vascular smooth muscle contractility due to a loss of smooth muscle α-actin decreases cellular mechanosensing by dysregulating cell adhesion to the matrix. Assessment of functional mechanical properties of the aorta by stress relaxation measurements in thoracic aortic rings suggested two functional regimes for Acta2−/− mice. Lower stress relaxation was recorded in aortic rings from Acta2−/− mice at tensions below 10 mN compared with wild type, likely driven by cytoskeletal-dependent contractility. However, no differences were recorded between the two groups above the 10 mN threshold, since at higher tension the matrix-dependent contractility may be predominant. In addition, our results showed that at any given level of stretch, transmural pressure is lower in aortic rings from Acta2−/− mice than wild type mice. In addition, a three-dimensional collagen matrix contractility assay showed that collagen pellets containing Acta2−/− smooth muscle cells contracted less than the pellets containing the wild type cells. Moreover, second harmonic generation non-linear microscopy revealed that Acta2−/− cells locally remodeled the collagen matrix fibers to a lesser extent than wild type cells. Quantification of protein fluorescence measurements in cells also showed that in absence of smooth muscle α-actin, there is a compensatory increase in smooth muscle γ-actin. Moreover, specific integrin recruitment at cell–matrix adhesions was reduced in Acta2−/− cells. Thus, our findings suggest that Acta2−/− cells are unable to generate external forces to remodel the matrix due to reduced contractility and interaction with the matrix. Impact statement Thoracic aneurysm formation is characterized by progressive enlargement of the ascending aorta, which predisposes the aorta to acute aortic dissection that can lead to sudden death. SMCs in the aorta play an integral role in regulating vessel wall contractility and matrix deposition in the medial layer. Recent studies show that mutations in genes associated with actomyosin apparatus reduce SMC contractility, increasing susceptibility to TAAD. Single-cell experiments enable discrete measurements of transient microscopic events that may be masked by a macroscopic average tissue behavior. Biophysical methods combined with microscopy techniques aid in understanding the specific roles of adhesion and cytoskeletal proteins in regulating SMC mechanosensing when SMα-actin is disrupted. Our findings suggest that Acta2− / − cells have increased SMγ-actin and decreased integrin recruitment at cell–matrix adhesion, hence a synthetic phenotype with reduced cellular mechanosensing.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 These authors contributed equally to the work.
ISSN:	1535-3702 1535-3699 1535-3699
DOI:	10.1177/1535370220903012