Identification of mutations in human smooth muscle actin {ACTA2) and myosin strongly suggest that disruption of smooth muscle cell contractile function predisposes individuals to thoracic aortic aneurysms and aortic dissections (TAAD). Project 1 seeks to determine the molecular mechanisms by which ACTA2 missense mutations lead to TAAD and occlusive vascular diseases. The hypothesis is that ACTA2 mutations lead to altered force output and energetic overload of smooth muscle cells, likely contributing to the eventual death of the cells. This proposal depends on our demonstrated ability to express human smooth muscle actin and myosin in Sf9 cells.
In Aim 1 we will assess if ACrA2 missense mutations alter filament assembly or the structural integrity of the filament. Single filament polymerization assays using total internal reflection fluorescence (TIRF) microscopy will be used to follow assembly and disassembly of individual filaments as a function of time, which will identify mutants with polymerization defects. The ability of the mutant actins to copolymerize with WT will be tested. Altered structural integrity of the filament will be evaluated by flexural rigidity measurements. Actin acts as a structural element through which force is transmitted, so even subtle alterations in the filament structure could have profound effects on force production by myosin.
In Aim 2 it will be determined if ACTA2 missense mutations affect the ability of myosin to generate motion and force. The interaction of actin with smooth muscle myosin will be assessed by an unloaded ensemble motility assay, which reflects the kinetics of the actomyosin interaction. Mutants will be further characterized under load using a force-clamp laser trap assay, which involves single actin filaments interacting with a small ensemble of myosin at different constant loads. These measurements will provide the average maximum isometric force generated by myosin. Decreased power output could trigger compensatory mechanisms in the smooth muscle cell. Homo and heteropolymers will be compared, as well as pure actin versus actin- tropomyosin.
Aim 3 determines if ACTA2 missense mutations alter actin filament structure, or the conformational dynamics of the filament. Actin filament structure will first be analyzed by electron microscopy of negatively stained filaments. High resolution cryo-electron microscopy (collaboration with Dr. Egelman) will then be used to test if the actin mutations interfere with the conformational dynamics of actin. This possibility is likely given that many of the mutations are buried, and cannot directly affect the interaction with myosin or other actin-binding proteins.

Public Health Relevance

Mutations in human smooth muscle actin (ACTA2) and myosin, and in the kinase that activates myosin, have been linked to thoracic aortic aneurysms (TAAD), as well as premature stroke and coronary artery disease. Mutations in ACTA2 are responsible for -15% of reported cases of TAAD. This project seeks to study the effect of the mutations in ACTA2 at the molecular level to allow an understanding of the pathological processes that take place, which in turn could lead to new insights and better treatments for these diseases.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
Project #
Application #
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Texas Health Science Center Houston
United States
Zip Code
Karimi, Ashkan; Milewicz, Dianna M (2016) Structure of the Elastin-Contractile Units in the Thoracic Aorta and How Genes That Cause Thoracic Aortic Aneurysms and Dissections Disrupt This Structure. Can J Cardiol 32:26-34
Wallace, S; Guo, D-C; Regalado, E et al. (2016) Disrupted nitric oxide signaling due to GUCY1A3 mutations increases risk for moyamoya disease, achalasia and hypertension. Clin Genet 90:351-60
Guo, Dong-chuan; Regalado, Ellen S; Gong, Limin et al. (2016) LOX Mutations Predispose to Thoracic Aortic Aneurysms and Dissections. Circ Res 118:928-34
Jondeau, Guillaume; Ropers, Jacques; Regalado, Ellen et al. (2016) International Registry of Patients Carrying TGFBR1 or TGFBR2 Mutations: Results of the MAC (Montalcino Aortic Consortium). Circ Cardiovasc Genet 9:548-558
Milewicz, Dianna; Hostetler, Ellen; Wallace, Stephanie et al. (2016) Precision medical and surgical management for thoracic aortic aneurysms and acute aortic dissections based on the causative mutant gene. J Cardiovasc Surg (Torino) 57:172-7
Abrams, Joshua; Einhorn, Zev; Seiler, Christoph et al. (2016) Graded effects of unregulated smooth muscle myosin on intestinal architecture, intestinal motility and vascular function in zebrafish. Dis Model Mech 9:529-40
Kuang, Shao-Qing; Medina-Martinez, Olga; Guo, Dong-Chuan et al. (2016) FOXE3 mutations predispose to thoracic aortic aneurysms and dissections. J Clin Invest 126:948-61
Chang, Audrey N; Kamm, Kristine E; Stull, James T (2016) Role of myosin light chain phosphatase in cardiac physiology and pathophysiology. J Mol Cell Cardiol 101:35-43
Ropars, Virginie; Yang, Zhaohui; Isabet, Tatiana et al. (2016) The myosin X motor is optimized for movement on actin bundles. Nat Commun 7:12456
Regalado, E S; Guo, D C; Santos-Cortez, R L P et al. (2016) Pathogenic FBN1 variants in familial thoracic aortic aneurysms and dissections. Clin Genet 89:719-23

Showing the most recent 10 out of 35 publications