Abstract 17193: Absence of Thoracic Aortic Disease In ACTA2R149C/+Mice is Associated With Defective Release of Mutant Smooth Muscle ?-actin From the Chaperonin Complex

Rationale:The most common mutation in ACTA2is p.Arg149Cys, which predisposes individuals to either thoracic aortic disease or premature onset coronary artery disease but rarely both diseases.Objective:We sought to identify the molecular mechanisms dictating whether or not an individual with ACTA2p.Arg149Cys has thoracic aortic disease.Methods and Results:A mouse model of the ACTA2p.Arg149Cys was generated using CRISPR/Cas9 technology (Acta2R149C/+). Acta2R149C/+mice have significantly decreased aortic contraction compared with wildtype (WT) mice (n=5 per group).Pathologic analysis shows significantly increased aortic medial wall thickness without evidence of medial degeneration (n=5 per group). The Acta2R149C/+mice do not form thoracic aortic aneurysms as assessed by echocardiography or experience sudden death when followed until 48 months of age (n=8 WT, n=7 Acta2R149C/+), and do not develop these complications when the blood pressure is increased by either exposure to a high salt diet and L-N;-nitroarginine methyl ester or trans-aortic constriction. In vitrostudies expressing WT and ACTA2mutants in Acta2-/-smooth muscle cells (SMCs) found decreased protein levels of R149C SM ?-actin when compared to WT SM ?-actin even though qPCR analyses showed the transcript levels were similar. Two dimensional gel electrophoresis studies confirmed reduced levels of the mutant compared with WT SM ?-actin in the cytoplasm in Acta2R149C/+SMCs. Actins are folded in the chaperonin containing t-complex polypeptide (CCT) folding complex, raising the possibility that the mutant actin may be released slower from this complex. Immunoprecipitation of the CCT complex using antibodies directed against CCT1, followed by immunoblot analyses for SM ?-actin, demonstrated increased SM ?-actin associated with the CCT complex in the Acta2R149C/+SMCs when compared to WT SMCs.Conclusions:These data indicate that the Acta2R149C/+mice do not develop thoracic aortic disease. The reduced amount of mutant SM ?-actin released from CCT chaperonin complex into the cytoplasm of aortic SMCs may contribute to the decreased penetrance of thoracic aortic disease in the Acta2R149C/+mouse model.