Hyaluronan 35 prevents endotoxin-mediated dysregulated skeletal muscle proteostasis during ethanol exposure

Sarcopenia in alcohol-related liver disease (ALD) is of high clinical significance, but there are no effective treatments. Hyaluronan 35 kDa (HA35), a glycosaminoglycan polymer, modulates responses to Toll-like receptor 4 (TLR4), a lipopolysaccharide receptor, to improve hepatic and macrophage function in ALD. We evaluated skeletal muscle responses to HA35 in preclinical models of ALD. Responses to ethanol, lipopolysaccharide (LPS), and HA35 were studied in differentiated murine C2C12/human-induced pluripotent stem cell (hiPSC)-derived myotubes, and wild-type/HA receptor CD44 knockout (CD44^-/-) mouse models of ALD (mALD). Signaling molecules and measures of protein homeostasis (proteostasis) were quantified by immunoblots, mitochondrial oxidative function was determined by high-sensitivity respirofluorometry, membrane elasticity by atomic force microscopy, and muscle contractile responses to electrical stimulation ex vivo were quantified. Multiomics, weighted gene co-expression network, and image analyses were also performed. Ethanol caused a sarcopenic phenotype in myotubes and mALD (lower myotube/muscle fiber diameter, muscle mass), less protein synthesis, impaired mTORC1 signaling, and higher autophagy markers and unaltered membrane elasticity. Expressions of LPS (TLR2, TLR4) and HA (CD44) receptors were upregulated by ethanol. In myotubes, LPS treatment caused a sarcopenic phenotype at lower concentrations in ethanol-treated myotubes. HA35 reversed ethanol/LPS-induced lower protein synthesis, impaired mTORC1 signaling, and mitochondrial complex I function in myotubes/muscle tissue. Muscle concentrations of HA fragments were lower in mALD, but beneficial responses to HA35 occurred without restoring HA concentrations, and HA35 was not beneficial in CD44^-/- mALD. Sarcopenia, signaling perturbations, and mitochondrial oxidative dysfunction in mALD are reversed by HA35, allowing for rapid clinical translation (ongoing clinicaltrials.gov, NCT05018481).NEW & NOTEWORTHY Sarcopenia in alcohol-related liver disease (ALD) is frequent and contributes to adverse clinical outcomes with limited treatment options. Hyaluronans (glycosaminoglycan polymers) of specific fragment sizes (30-40 kDa) have anti-inflammatory and tissue-protective properties. In an array of preclinical models of ALD, we demonstrate that HA35 reverses signaling, mitochondrial oxidative function, and phenotypic perturbations associated with sarcopenia in ALD. These effects are mediated without restoring the low muscle tissue concentrations of HA in ALD.