WNT3a induces external anal sphincter fibrosis and dysfunction in rabbits

Injury and aging of the external anal sphincter (EAS) muscle lead to fibrosis and muscle dysfunction, major contributors to fecal incontinence. Activation of the WNT/β-catenin signaling pathway has been linked to fibrosis in various tissues, including skeletal muscle. This study examined whether the WNT agonist Wnt3a induces fibrosis and dysfunction in the EAS muscle. Adult female New Zealand White rabbits received four local injections of Wnt3a or saline into the EAS muscle. Anal canal pressure was measured by manometry every 2 wk for 8 wk, followed by histological, immunofluorescent, immunohistochemistry (IHC), Western blot, and proteomic analyses of the EAS muscle. Rabbits treated with Wnt3a exhibited a significant reduction in anal canal pressure 8 wk postinjection (P ≤ 0.05) compared with controls. Histologic evaluation revealed increased connective tissue (P = 0.06), significant collagen deposition, and decreased muscle area and fiber thickness (P ≤ 0.05). Western blot analysis showed elevated levels of β-catenin, nuclear active β-catenin (PY489), Smad1/2/3, signal transducer and activator of transcription 3 (Stat3), transforming growth factor-β (TGF-β), and vimentin (P ≤ 0.05), with p-Stat3, p-Smad3, and collagen-4 trending upward. Immunofluorescence and IHC confirmed increased β-catenin, collagen-4, and TGF-β levels, and proteomic data indicated altered pathways related to muscle contraction, fibrosis, and atrophy. These findings demonstrate that direct administration of a WNT agonist promotes EAS fibrosis and dysfunction, mirroring changes associated with aging and injury. Local application of WNT antagonists may represent a therapeutic strategy to prevent anal sphincter dysfunction following injury.NEW & NOTEWORTHY In the current study, for the first time we investigated the effects of a local injection of Wnt3a-an agonist of the WNT signaling pathway-on EAS function, muscle replacement by fibrosis, and the activation of downstream WNT signaling pathways. Wnt3a injection resulted in impaired EAS function and replacement of muscle with fibrosis. Notably, the downstream signaling remained active even 8 wk after the Wnt3a injection.