Introduction

Patients with end-stage liver disease (ESLD) often present with sarcopenia, defined as loss of skeletal muscle mass and quality, which is associated with reduced quality of life and increased mortality. However, the molecular mechanisms driving sarcopenia in ESLD are not fully understood and there are currently no therapeutic interventions.

 

Aims and Objectives

This study aimed to identify potential circulating factors that may contribute to sarcopenia progression in ESLD, through driving transcriptomic changes in the skeletal muscle. To achieve this, we had 3 main objectives:

 

1.Profile the skeletal muscle transcriptome of patients with ESLD and age and sex matched healthy controls (HC) to identify potential mechanism of muscle dysfunctions in ESLD.

 

2 Determine whether the altered skeletal muscle transcriptome in ESLD may be driven by circulating factors, by stimulating primary human myotubes with ESLD patient plasma and performing RNAseq.

 

3. Profile patient serum to identify differential cytokines in ESLD and cross-reference with skeletal muscle transcriptomic data to identify potential candidates driving altered gene expression.

 

Methods
Quadriceps muscle tissue, plasma and serum was obtained from ESLD patients (n=24) and age/sex-matched HC (n=18). ESLD patients were recruited to a larger prospective observational study, The Evaluation of Sarcopenia in Inflammatory Disease (clinical trial ID: NCT04734496, ethical approval 18/WM/0167). Local ethical approval was granted for recruitment of HC (ERN_19-0831). This study was conducted in accordance with the Declaration of Helsinki. Total RNA was isolated from snap frozen muscle tissue, obtained via muscle biopsy of the vastus lateralis, and subjected to RNAseq (Illumina). Serum levels of 62 cytokines were profiled by Luminex and ELISA. In vitro, primary human myotubes were cultured with media containing 10% ESLD, or HC plasma (24h, n=6) followed by RNAseq (BGI genomics). Differentially expressed genes (p<0.05, fold-change >1.5) were determined using Qlucore and DESeq2, with subsequent pathway analysis performed utilising Ingenuity (IPA, Qiagen). Statistically significant cytokines were determined by either Student's t tests or Mann-Whitney U tests as appropriate.

 

Results
387 and 225 genes were significantly up- and downregulated respectively in ESLD muscle compared to HC, with cellular senescence identified as a top dysregulated cellular function by IPA. Upstream regulators predicted to drive these transcriptomic changes in ESLD included hepatocyte growth factor (HGF) and interleukin-1 signalling. Serum levels of 16 cytokines were significantly (p<0.05) greater and 4 significantly lower (p<0.05) in ESLD, including HGF and interleukin-1 receptor antagonist respectively. Treatment of myotubes with ESLD plasma partly replicated the transcriptomic phenotype of ESLD muscle, with significant activation of cellular senescence pathways observed and interleukin-1 again a purported upstream regulator.

 

Conclusions
In conclusion, skeletal muscle of ESLD patients exhibits an altered transcriptome associated with increased cellular senescence, which may be partly driven by circulating inflammatory mediators, including HGF and IL-1. Targeting such mediators may provide a novel therapeutic intervention to limit sarcopenia progression in ESLD patients.