Myosin Light Chain 9 Mediates Graft Fibrosis After Pediatric Liver Transplantation Through TLR4/MYD88/NF-κB Signaling.

Abstract

Background & aims: The incidence of graft fibrosis is elevated after pediatric liver transplantation (pLT) and is influenced by cold ischemic time (CIT). Myosin light chain 9 (MYL9), a member of the myosin family, could act on hepatic stellate cells (HSCs) and induce a transition to active phase. We hypothesized that cold ischemic injury could stimulate MYL9 expression and lead to graft fibrosis.

Methods: We tested the hypothesis by analyzing multi-omics data from human protocol liver biopsy samples 2 years after LT, performing rat LT with different CIT and conducting in vitro studies in HSC cell lines with MYL9 knockdown and overexpression.

Results: Clinically, CIT is an independent risk factor for graft fibrosis after pLT. Omics analysis identified MYL9 as a prominent contributor in graft fibrosis. MYL9 is strongly correlated with liver fibrosis grade and the progression of fibrosis. The study of rat LT model demonstrated MYL9 expression increases with the prolongation of CIT, and its role is specific to transplant setting. Mechanistically, in vitro experiments with HSCs exposed to hypoxia/reoxygenation revealed a substantial decrease in HSCs activation after MYL9 knockdown. Conversely, overexpression of MYL9 significantly enhanced the activation of HSCs. Subsequent transcriptome sequencing of HSCs with MYL9 knockdown unveiled that MYL9 primarily functions through the TLR4/MYD88/NF-κB signaling pathway. Liver graft fibrosis was ameliorated when toll like receptor 4 signaling was inhibited in rats.

Conclusions: Our findings demonstrate that prolonged CIT up-regulates the expression of MYL9 in liver graft after LT. MYL9 activates HSCs and promotes fibrosis through a TLR4/MYD88/NF-κB signaling dependent manner.