Abstract Body: Introduction: Spontaneous coronary artery dissection (SCAD) is the cause of 1.7-4% of acute coronary syndromes (ACS), with a strongly sex-biased prevalence (90% women). In a whole exome sequencing study of SCAD, we recently reported exonic variants in ADAMTSL4 that were predicted to be deleterious by in silico analysis. We and others have previously reported a robustly associated common variant, rs12740679 at chromosome 1q21.2, identified by genome-wide associations study of SCAD that demonstrated a colocalized expression quantitative trait locus effect on ADAMTSL4, with reduced gene expression associated with the SCAD risk allele.

Methods: Secretion assays were performed using human 293T cells transfected with overexpression constructs coding ADAMTSL4 WT, or ADAMTSL4 c.2263dupG (p.Gly758Trpfs*59) and ADAMTSL4 c.1249C>T (p.Arg417Cys). Female human coronary artery smooth muscle cells (huCASMCs) from 3-6 individual tissue donors were transfected using ADAMTSL4 siRNA or scramble control siRNA molecules. In vitro huCASMCs were evaluated by bulk-RNAseq, MS-proteomics, microfibril assays, migration assays, traction force microscopy (TFM), immunocytochemistry (ICC), and 3D arterial ring organoid (ARO) tensile testing.

Results: In vitro secretion assays showed a significant 50% decrease in ADAMTSL4 protein detected by western blot in the conditioned media of cells transfected with both variant ADAMTSL4 constructs when compared to ADAMTSL4 WT. RNAseq and mass-spectroscopy proteomics comparing scramble control siRNA to ADAMTSL4 siRNA-treated huCASMCs showed effects on ECM maturation/stability, as well as cell adhesion and migration. Cellular microfibril assays showed a significant decrease in ADAMTSL4 binding and fibrillogenesis when cells were cultured in the ADAMTSL4 variant-conditioned media. ADAMTSL4 knock-down huCASMCs showed significantly increased cell adhesion and migration, and decreased fibrillogenesis as well. The number of vinculin focal adhesions, via ICC staining, was increased in cells with decreased ADAMTSL4 expression, and gamma-actin protein expression was increased. AROs created with ADAMTSL4 knock down huCASMCs, showed a significant decrease in ultimate tensile strength, elastic modulus, and failure strength.

Conclusions: These findings support a role for ADAMTSL4 in arterial smooth muscle and suggest that ADAMTSL4 loss may cause abnormal cellular and extracellular effects which can lead to arterial destabilization in SCAD.