Abstract Tu0026: A Mechanistic Insight Into The Connection Between Metabolism And Differentiation In ACTA2 P. R179 Smooth Muscle Cells

Genetic variants in ACTA2 altering arginine 179 cause Smooth Muscle Dysfunction Syndrome, which presents with thoracic aortic disease, moyamoya-like cerebrovascular disease, and other vascular complications in childhood. Explanted Acta2 R179C/+ smooth muscle cells (SMCs) are less differentiated than wildtype (WT) SMCs. Acta2 R179C/+ SMCs fail to fully differentiate and retain stem cell phenotypes, including relying on glycolysis rather than oxidative phosphorylation (OXPHOS) for compensatory production of ATP. When treated with nicotinamide riboside (NR), Acta2 R179C/+ SMCs decrease glycolysis and increase OXPHOS, but also decrease migration and increase contractile SMCs markers, suggesting a link between metabolism and SMC differentiation. Metabolomic experiments using 1,2- 13 C 2 -glucose tracer show a higher accumulation of TCA cycle metabolites such as 2-hydroxyglutarate-succinic acid (2HG) in Acta2 R179C/+ SMCs. NR treatment decreases flux through glycolysis and TCA cycle metabolites in both WT and Acta2 R179C/+ SMCs, including significant reductions in 2HG. TCA cycle metabolite 2HG has been characterized as a competitive inhibitor of α-KG-dependent dioxygenases, including histone demethylases such as Ten-eleven translocation 2 (TET2). TET2 is a DNA demethylase, which oxidizes 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) leading to DNA demethylation and gene activation, and TET2 was previously identified as a master regulator of SMC phenotype. At baseline, TET2 expression is lower in Acta2 R179C/+ SMCs compared to WT, and treatment with NR increases TET2 levels in both cell lines. Additionally, chromatin immunoprecipitation (ChIP) assays show decreased TET2 binding to SMC loci in mutant cells, which is rescued by NR treatment. Hydroxy methylated DNA immunoprecipitations show lower 5-hmC levels on SMC promoters in mutant cells compared to WT, and 5-hmC is increased in both cell lines after NR treatment. To determine if these findings represent a generalizable mechanism linking SMC metabolism with differentiation, WT SMCs were treated with 2HG, leading to a decrease in contractile protein and TET2 levels. Further, the addition of exogenous 2HG prevents NR from increasing contractile protein levels in Acta2 R179C/+ SMCs. These findings suggest that NR increases SMC differentiation by preventing the accumulation of 2HG, releasing the inhibition of TET2, and allowing for the demethylation of contractile gene promoters, thus rescuing SMC phenotype.