Abstract Thu145: Investigating the Regulatory Roles of SERTAD4 in Cardiac Fibroblast Activation

Cardiac disease remains the leading cause of death nationally and globally. Moreover, cardiac remodeling, particularly scar tissue formation known as cardiac fibrosis facilitated by activated cardiac fibroblasts (CFs), is well associated with heart failure. One major contributing signaling pathway to CF activation, the Transforming Growth Factor – Beta (TGF-β) pathway has been targeted for treatment against cardiac fibrosis. Unfortunately, obvious therapeutic strategies for this pathway have resulted in on-target toxicity due to pleiotropic roles for this pathway (most importantly maintenance of differentiation states). This necessitates uncovering downstream players in cardiac fibroblast activation that won’t perturb cardiac processes and beyond. A downstream protein upregulated by BRD4 post-TGF-β treatment known as Serta domain-containing protein 4 (SERTAD4) was an identified as potential regulator of CF activation. Global SERTAD4 knockout mice are protected from post-ischemic and nonischemic injuries (preserved systolic cardiac function). We’ve also shown that knocking out SERTAD4 appears to reduce fibrotic remodeling in cardiac-injured mice and in aging mice. Thus, we hypothesized that SERTAD4 plays key regulatory roles in cardiac fibroblast activation through transcriptional coactivating mechanisms. We test this hypothesis by 1) determining what CF behaviors SERTAD4 modulates and 2) identifying what SERTAD4 protein-protein interactions facilitate CF activation. We investigated CF behaviors by exposing Adult Rat Ventricular Fibroblasts (ARVFs) to siRNA and shRNA knockdown regimes targeting SERTAD4 and conducting cellular assays that determine proliferation, migration, and differentiation capacities. Additionally, we utilized a dual, immunoprecipitation scheme using epitope-tagged SERTAD4 constructs to determine protein-protein interactions and close-proximity labeling with TurboID-fused SERTAD4 constructs to determine live-cell closeness of SERTAD4 with other proteins. We found that knocking down the same SERTAD4 mRNA transcripts with either siRNA or shRNA approaches leads to varying outcomes in TGF-β signaling, leading to decreased proliferative and differentiative capacity, though variable migratory behavior. From our IP studies, we determine potential interactions between SERTAD4 and PP2A subunits.