Abstract Body: Smooth muscle cell (SMC) phenotypic modulation is a hallmark of atherosclerotic plaque progression and stability. Among disease-associated SMC states, fibromyocytes play a key protective role in stabilizing the fibrous cap. Although alternative splicing (AS) is known to regulate SMC dedifferentiation, the AS isoforms driving SMC fibromyocyte differentiation in human coronary artery disease remain uncharacterized. This study tests the hypothesis that distinct alternative splicing programs underlie phenotypic transitions from contractile to fibromyocyte SMCs in atherosclerosis.
Bulk short read RNAseq data from human coronary arteries (CA) was analyzed with rMATS to identify lesion-associated AS events (lesion n=31; non-lesion n=29). In parallel, immortalized human coronary artery SMCs (iHCASMCs) were induced toward a fibromyocyte phenotype using PDGF-BB and TGF-β1 (n=3 biological replicates) and profiled by PacBio long read RNA sequencing. CA AS events were mapped onto iHCASMC long read transcripts, and predicted effects on protein interactions were assessed using NEASE. Primary outcomes included differential splicing quantification and network-level perturbation.
Across coronary arteries, 189,075 differential AS events were identified in 15,788 genes. 61% (115,822 of 189,075) of AS events mapped to full-length transcripts detected in basal and fibromyocyte iHCASMCs. Investigating established SMC subtype markers revealed that 90% (90 of 100) of established fibromyocyte marker genes harbored at least one mapped event. The set of transcript-mapped events also recapitulated established atherosclerotic VSMC splicing events, including ACTN1 exon 19a/19b (ΔPSI –0.091, FDR<0.05) and truncation of vinculin (VCL) tail domain in fibromyocytes (ΔPSI 0.057, FDR<0.05). 17.4% of genes (2083 of 11,955) harbored predicted disruptions to protein domains and interactions; splice-aware pathway analysis indicated cytoskeletal folding and chaperonin pathways, particularly the CCT/TriC complex, were most enriched for splicing-associated changes based on associated genes' impacts.
These data indicate that fibromyocyte differentiation in atherosclerosis is governed by coordinated alternative splicing programs that affect cytoskeletal and proteostasis networks. This work establishes a blueprint for characterizing SMC phenotypic remodeling in coronary artery disease and extending these analyses to additional disease-relevant SMC states.