THU571 Mechanisms Of Estrogen-induced Skeletal Muscle Fibrosis In An Inguinal Hernia Humanized Aromatase Mouse Model

Disclosure: T. Potluri: None. H. Zhao: None. P. Yin: None. J.J. Stulberg: None. R.L. Lieber: None. S.E. Bulun: None. Mechanisms of Estrogen-Induced Skeletal Muscle Fibrosis in an Inguinal Hernia Humanized Aromatase Mouse Model Abdominal skeletal muscle weakening via fibrosis and atrophy leads to inguinal herniation. Despite the high prevalence and morbidity of inguinal herniation, the mechanisms and etiology are largely unknown. We developed a transgenic mouse model with the human aromatase gene (Arom(hum)), where all male mice develop spontaneous scrotal hernias at puberty. The lower abdominal muscles (LAM) in these mice can synthesize estradiol (E2), which activates the stromal fibroblasts, leading to fibrosis and herniation. Remarkably, the administration of Estrogen Receptor Alpha (ERα) antagonist Fulvestrant to the herniated mice reverses fibrosis and herniation. We use this novel model to uncover mechanisms of E2-induced skeletal muscle fibrosis and its subsequent reversal. Hernia-Associated Fibroblasts (HAFs) were isolated from LAM tissues and cultured under estrogen-replete (10nM E2) and deplete (10nM E2 + 100nM Fulvestrant) conditions. The cells were analyzed using ERα ChIP-seq, ATAC-seq, and RNA-seq to observe genetic, epigenetic, and transcriptomic changes (n = 10-15 mice/group in 3 technical replicates).Via RNA-seq, we identified 853 differentially expressed genes between E2 and E2+Fulvestrant treated fibroblasts. These genes were associated with the extracellular matrix, WNT signaling, TGFβ, and focal adhesions were enriched in our study. We further identified 657 E2-upregulated annotated genes common between ATAC-seq and ChIP-seq. Overall, 58 genes were identified across all three datasets, including known E2-responsive genes such as Pgr, as well as extracellular modulators such as Adamts3, Adamts6, Pcdh7, Ltbp1, Fbln5, and Fbln7. Furthermore, E2-ERα activation also directly induced the expression of other transcription regulators, such as Pbx1, Id2, Hlf, Zfp521, and Zfp618. Together, we identified the core E2-modulated genes in HAFs that lead to skeletal muscle fibrosis and reversal. To validate these findings, we performed RNAscope on human inguinal hernia tissues. We detected PBX1, PIEZO2, NRP2, LTBP1, NCAM1, and ADAMTS6 expression in the herniated fibrotic regions of the samples. In contrast, there is no expression of these genes in corresponding healthy muscle fibers.Using this Arom(hum) mouse model, we characterized, for the first time, characterize E2 mechanisms in skeletal muscle fibrosis and herniation. In addition, we provide an insight into the specific disease-causing fibroblast populations and markers that can form the source for future therapeutics to treat hernias and other fibrotic disorders. Presentation Date: Thursday, June 15, 2023