ABSTRACT: Background. Skeletal muscle is a major target organ for ethanol induced perturbations with resultant sarcopenia in alcohol-related liver disease (ALD). Targeted studies show that in skeletal muscle, ethanol causes mitochondrial oxidative dysfunction and impaired mTORC1 signaling with consequent decreased protein synthesis and increased autophagy. However, complex interactions and pathway intersections involved in cellular adaptive and maladaptive responses are not well understood. Unlike hypotheses driven focused experiments, an integrated multiomics-experimental validation approach helps identify the global landscape of complex, interacting perturbations in the skeletal muscle. Methods. We used an integrated multiomics approach in a comprehensive array of models, including murine and human induced pluripotent stem cell-derived myotubes (hiPSCm), skeletal muscle from a mouse model of ALD (mALD), and human patients with alcohol-related cirrhosis (CIR) and controls (CON), to identify novel regulatory mechanisms of sarcopenia in ALD. We generated 13 untargeted datasets, including chromosomal conformation, RNA sequencing, proteomics, phosphoproteomics, acetylomics, and metabolomics and conducted a comprehensive analysis using upset plots and feature extraction-based approaches. The most altered molecular interactions were experimentally validated in different models using immunoblots, redox measurements, imaging methods, and measures of senescence associated molecular phenotype (SAMP). Redox ratio was reversed using mitochondrial targeted Lactobacillus brevis NADH oxidase (MitoLbNOX). Results. Enrichments in mitochondrial oxidative function, protein synthesis, and senescence pathways were identified. Multiomics analyses were consistent with the known effects of hypoxia inducible factor 1 (HIF1) that inhibits mitochondrial oxidative dysfunction and promotes skeletal muscle senescence. Pertubrations in genes in the nicotinamide dinucleotide-related pathways including sirtuin signaling, known regulators of senescence, was also noted with ethanol treatment. Experimentally, HIF1 protein was stabilized in myotubes and muscle tissue without cellular hypoxia. Mitochondrial electron transport chain protein expression was less in murine myotubes and hiPSCm with lower redox ratio, less expression of sirtuins, and increased acetylation of mitochondrial proteins. SAMP noted in multiple models in response to ethanol exposure and reversing redox ratio with MitoLbNOX prevented HIF1a stabilization and SAMP in murine myotubes. Conclusions. Our integrated multiomics analyses combined with complementary experimental validation, identify HIF1a stabilization with accelerated skeletal muscle post-mitotic senescence as novel mechanisms of sarcopenia in ALD, highlighting the complex interactions that result in mitochondrial dysfunction with persistent and progressive sarcopenia in ALD.
Ling Li 
