Sarcopenia, a condition of low muscle mass, quality, and strength, is commonly found in patients with cirrhosis and is associated with adverse clinical outcomes including reduction in quality of life, increased mortality, and posttransplant complications. In chronic liver disease (CLD), sarcopenia is most commonly defined through the measurement of the skeletal muscle index of the third lumbar spine. A major contributor to sarcopenia in CLD is the imbalance in muscle protein turnover, which likely occurs due to a decrease in muscle protein synthesis and an elevation in muscle protein breakdown. This imbalance is assumed to arise due to several factors including accelerated starvation, hyperammonemia, amino acid deprivation, chronic inflammation, excessive alcohol intake, and physical inactivity. In particular, hyperammonemia is a key mediator of the liver-gut axis and is known to contribute to mitochondrial dysfunction and an increase in myostatin expression. Currently, the use of nutritional interventions such as late-evening snacks, branched-chain amino acid supplementation, and physical activity have been proposed to help the management and treatment of sarcopenia. However, little evidence exists to comprehensively support their use in clinical settings. Several new pharmacological strategies, including myostatin inhibition and the nutraceutical Urolithin A, have recently been proposed to treat age-related sarcopenia and may also be of use in CLD. This review highlights the potential molecular mechanisms contributing to sarcopenia in CLD alongside a discussion of existing and potential new treatment strategies.

In vitro models of muscle aging are useful for understanding mechanisms of age-related muscle loss and aiding the development of targeted therapies. To investigate mechanisms of age-related muscle loss in vitro utilizing ex vivo human serum, fasted blood samples were obtained from four old (72 ± 1 yr) and four young (26 ± 3 yr) men. Older individuals had elevated levels of plasma CRP, IL-6, HOMA-IR, and lower concentric peak torque and work-per-repetition compared with young participants (P < 0.05). C2C12 myotubes were serum and amino acid starved for 1 h and conditioned with human serum (10%) for 4 h or 24 h. After 4 h, C2C12 cells were treated with 5 mM leucine for 30 min. Muscle protein synthesis (MPS) was determined through the surface sensing of translation (SUnSET) technique and regulatory signaling pathways were measured via Western blot. Myotube diameter was significantly reduced in myotubes treated with serum from old, in comparison to young donors (84%, P < 0.001). MPS was reduced in myotubes treated with old donor serum, compared with young serum before leucine treatment (32%, P < 0.01). MPS and the phosphorylation of Akt, p70S6K, and eEF2 were increased in myotubes treated with young serum in response to leucine treatment, with a blunted response identified in cells treated with old serum (P < 0.05). Muscle protein breakdown signaling pathways did not differ between groups. In summary, we show that myotubes conditioned with serum from older individuals had decreased myotube diameter and MPS compared with younger individuals, potentially driven by low-grade systemic inflammation.

In vitro models provide an important platform for the investigation of cellular growth and atrophy to inform, or extend mechanistic insights from, logistically challenging in vivo trials. Although these models allow for the identification of candidate mechanistic pathways, many models involve supraphysiological dosages, nonphysiological conditions, or experimental changes relating to individual proteins or receptors, all of which limit translation to human trials. To overcome these drawbacks, the use of ex vivo human plasma and serum has been used in cellular models to investigate changes in myotube hypertrophy, cellular protein synthesis, anabolic and catabolic markers in response to differing age, disease states, and nutrient status. However, there are currently no concurrent guidelines outlining the optimal methodology for this model. This review discusses the key methodological considerations surrounding the use of ex vivo plasma and serum with a focus in application to skeletal muscle cell lines (i.e., C2C12, L6, and LHCN-M2) and human primary skeletal muscle cells (HSMCs) as a means to investigate molecular signaling in models of atrophy and hypertrophy, alongside future directions.

Cocoa flavanols have been shown to improve muscle function and may offer a novel approach to protect against muscle atrophy. Hippuric acid (HA) is a colonic metabolite of (-)-epicatechin (EPI), the primary bioactive compound of cocoa, and may be responsible for the associations between cocoa supplementation and muscle metabolic alterations. Accordingly, we investigated the effects of EPI and HA upon skeletal muscle morphology and metabolism within an in vitro model of muscle atrophy. Under atrophy-like conditions (24h 100μM dexamethasone (DEX)), C2C12 myotube diameter was significantly greater following co-incubation with either 25μM HA (11.19±0.39μm) or 25μM EPI (11.01±0.21μm) compared to the vehicle control (VC; 7.61±0.16μm, both P < .001). In basal and leucine-stimulated states, there was a significant reduction in myotube protein synthesis (MPS) rates following DEX treatment in VC (P = .024). Interestingly, co-incubation with EPI or HA abrogated the DEX-induced reductions in MPS rates, whereas no significant differences versus control treated myotubes (CTL) were noted. Furthermore, co-incubation with EPI or HA partially attenuated the increase in proteolysis seen in DEX-treated cells, preserving LC3 α/β II:I and caspase-3 protein expression in atrophy-like conditions. The protein content of PGC1α, ACC, and TFAM (regulators of mitochondrial function) were significantly lower in DEX-treated versus. CTL cells (all P < .050). However, co-incubation with EPI or HA was unable to prevent these DEX-induced alterations. For the first time we demonstrate that EPI and HA exert anti-atrophic effects on C2C12 myotubes, providing novel insight into the association between flavanol supplementation and favourable effects on muscle health.

Sarcopenia is a common complication affecting liver disease patients, yet the underlying mechanisms remain unclear. We aimed to elucidate the cellular mechanisms that drive sarcopenia progression using an in vitro model of liver disease. C2C12 myotubes were serum and amino acid starved for 1-h and subsequently conditioned with fasted ex vivo serum from four non-cirrhotic non-alcoholic fatty liver disease patients (NAFLD), four decompensated end-stage liver disease patients (ESLD) and four age-matched healthy controls (CON) for 4- or 24-h. After 4-h C2C12 myotubes were treated with an anabolic stimulus (5 mM leucine) for 30-min. Myotube diameter was reduced following treatment with serum from ESLD compared with CON (−45%) and NAFLD (−35%; p < 0.001 for both). A reduction in maximal mitochondrial respiration (24% and 29%, respectively), coupling efficiency (~12%) and mitophagy (~13%) was identified in myotubes conditioned with NAFLD and ESLD serum compared with CON (p < 0.05 for both). Myostatin (43%, p = 0.04) and MuRF-1 (41%, p = 0.03) protein content was elevated in myotubes treated with ESLD serum compared with CON. Here we highlight a novel, experimental platform to further probe changes in circulating markers associated with liver disease that may drive sarcopenia and develop targeted therapeutic interventions.