In vitro skeletal muscle cell atrophy and mitochondrial dysfunction after exposure to human serum from patients with ESLD and NAFLD

Background and aims: Sarcopenia, defined as a loss of muscle mass, strength and function is a common complication associated with End Stage Liver Disease (ESLD). Despite its clinical importance, the mechanisms which contribute to the development of sarcopenia in ESLD remain unclear. Therefore, we aimed to investigate the mechanistic drivers of sarcopenia in ESLD through the use of an in vitro model utilising ex vivo human serum from ESLD and non alcoholic fatty liver disease (NAFLD) patients.

Method: Fasted blood samples were obtained from 4 age-matched control (64.7±10.0), 4 non-cirrhotic NAFLD (61.8±7.6) and 4 decompensated ESLD (60.5±1.7) patients. C2C12 myotubes were serum and amino acid starved for 1 hour and subsequently conditioned with serum from control, NAFLD and ESLD patients for 4 or 24 hours. After 4 hours C2C12 myotubes were treated with 5 mM leucine for 30-minutes. Myotube diameter was assessed using fluorescent microscopy. Mitochondrial function was measured through: (1) respiration, utilizing a mitochondrial stress test on a XFe24 Seahorse Analyser; (2) mitophagy, assessed using a C2C12 mitoQC cell line. Muscle protein synthesis (MPS) was determined using the surface sensing of translation (SUnSET) technique and signaling markers of anabolic and catabolic pathways was measured via Western blotting.

Results: Myotube diameter was reduced in myotubes treated with serum from ESLD patients in comparison to both control (45%) and NAFLD (35%) patients (p<0.001 for both). A reduction in maximal mitochondrial respiration, coupling efficiency and mitophagy was identified in myotubes conditioned with both NAFLD and ESLD serum in comparison to controls (p<0.05 for both). No difference in mitochondrial protein content was identified. MuRF-1, a catabolic protein, was elevated in myotubes treated with ESLD serum in comparison to control serum (p=0.03). Leucine treatment did not alter muscle protein breakdown (MPB) signaling in any group. No change in MPS or anabolic signaling markers was identified within or between groups, in the presence or absence of leucine treatment.

Conclusion: We show myotube atrophy in cells conditioned with ex vivo human serum from ESLD patients in comparison to control and NAFLD patients, which may be caused through mitochondrial dysfunction and an increase in MPB. This work provides an experimental platform to further probe mechanisms of sarcopenia within ESLD and to develop potential therapeutic targets to mitigate sarcopenia.