Loss of muscle mass, or sarcopenia, is nearly universal in cirrhosis and adversely affects patient outcome. The underlying cross-talk between the liver and skeletal muscle mediating sarcopenia is not well understood. Hyperammonemia is a consistent abnormality in cirrhosis due to impaired hepatic detoxification to urea. We observed elevated levels of ammonia in both plasma samples and skeletal muscle biopsies from cirrhotic patients compared with healthy controls. Furthermore, skeletal muscle from cirrhotics had increased expression of myostatin, a known inhibitor of skeletal muscle accretion and growth. In vivo studies in mice showed that hyperammonemia reduced muscle mass and strength and increased myostatin expression in wild-type compared with postdevelopmental myostatin knockout mice. We postulated that hyperammonemia is an underlying link between hepatic dysfunction in cirrhosis and skeletal muscle loss. Therefore, murine C2C12 myotubes were treated with ammonium acetate resulting in intracellular concentrations similar to those in cirrhotic muscle. In this system, we demonstrate that hyperammonemia stimulated myostatin expression in a NF-κB-dependent manner. This finding was also observed in primary murine muscle cell cultures. Hyperammonemia triggered activation of IκB kinase, NF-κB nuclear translocation, binding of the NF-κB p65 subunit to specific sites within the myostatin promoter, and stimulation of myostatin gene transcription. Pharmacologic inhibition or gene silencing of NF-κB abolished myostatin up-regulation under conditions of hyperammonemia. Our work provides unique insights into hyperammonemia-induced myostatin expression and suggests a mechanism by which sarcopenia develops in cirrhotic patients.signaling | portosystemic shunting

Patients with alcoholic cirrhosis and hepatitis have severe muscle loss. Since ethanol impairs skeletal muscle protein synthesis but does not increase ubiquitin proteasome-mediated proteolysis, we investigated whether alcohol-induced autophagy contributes to muscle loss. Autophagy induction was studied in: A) Human skeletal muscle biopsies from alcoholic cirrhotics and controls, B) Gastrocnemius muscle from ethanol and pair-fed mice, and C) Ethanol-exposed murine C2C12 myotubes, by examining the expression of autophagy markers assessed by immunoblotting and real-time PCR. Expression of autophagy genes and markers were increased in skeletal muscle from humans and ethanol-fed mice, and in myotubes following ethanol exposure. Importantly, pulse-chase experiments showed suppression of myotube proteolysis upon ethanol-treatment with the autophagy inhibitor, 3-methyladenine (3MA) and not by MG132, a proteasome inhibitor. Correspondingly, ethanol-treated C2C12 myotubes stably expressing GFP-LC3B showed increased autophagy flux as measured by accumulation of GFP-LC3B vesicles with confocal microscopy. The ethanol-induced increase in LC3B lipidation was reversed upon knockdown of Atg7, a critical autophagy gene and was associated with reversal of the ethanol-induced decrease in myotube diameter. Consistently, CT image analysis of muscle area in alcoholic cirrhotics was significantly reduced compared with control subjects. In order to determine whether ethanol per se or its metabolic product, acetaldehyde, stimulates autophagy, C2C12 myotubes were treated with ethanol in the presence of the alcohol dehydrogenase inhibitor (4-methylpyrazole) or the acetaldehyde dehydrogenase inhibitor (cyanamide). LC3B lipidation increased with acetaldehyde treatment and increased further with the addition of cyanamide. We conclude that muscle autophagy is increased by ethanol exposure and contributes to sarcopenia.

The mechanism of the nearly universal decreased muscle strength in cirrhosis is not known. We evaluated whether hyperammonemia in cirrhosis causes contractile dysfunction independent of reduced skeletal muscle mass. Maximum grip strength and muscle fatigue response were determined in cirrhotic patients and controls. Blood and muscle ammonia concentrations and grip strength normalized to lean body mass were measured in the portacaval anastomosis (PCA) and sham-operated pair-fed control rats (n = 5 each). Ex vivo contractile studies in the soleus muscle from a separate group of Sprague-Dawley rats (n = 7) were performed. Skeletal muscle force of contraction, rate of force development, and rate of relaxation were measured. Muscles were also subjected to a series of pulse trains at a range of stimulation frequencies from 20 to 110 Hz. Cirrhotic patients had lower maximum grip strength and greater muscle fatigue than control subjects. PCA rats had a 52.7 ± 13% lower normalized grip strength compared with control rats, and grip strength correlated with the blood and muscle ammonia concentrations (r(2) = 0.82). In ex vivo muscle preparations following a single pulse, the maximal force, rate of force development, and rate of relaxation were 12.1 ± 3.5 g vs. 6.2 ± 2.1 g; 398.2 ± 100.4 g/s vs. 163.8 ± 97.4 g/s; -101.2 ± 22.2 g/s vs. -33.6 ± 22.3 g/s in ammonia-treated compared with control muscle preparation, respectively (P < 0.001 for all comparisons). Tetanic force, rate of force development, and rate of relaxation were depressed across a range of stimulation from 20 to 110 Hz. These data provide the first direct evidence that hyperammonemia impairs skeletal muscle strength and increased muscle fatigue and identifies a potential therapeutic target in cirrhotic patients.

Background and Aims Sarcopenia or skeletal muscle loss adversely affects outcomes in cirrhosis. The impact of aetiology of liver disease on the severity or the rate of muscle loss is not known. Methods Consecutive, well‐characterized adult patients with cirrhosis due to viral hepatitis (VH), alcoholic liver disease (ALD) or non‐alcoholic fatty liver disease (NAFLD) and non‐diseased controls with at least two temporally distinct abdominal CT (computed tomography) scans were evaluated. Psoas, paraspinal and abdominal wall muscle areas at the L3 vertebra level were quantified on the CT scans. Standardized rate of change in muscle area was expressed as change in area/100 days. Univariate and multivariable analyses were performed to identify contributors to rate of muscle loss and survival. Results Among 83 cirrhotics (NAFLD n = 26, ALD n = 39, VH n = 18), there were 20 (24.1%) deaths over 62.7 ± 41.3 months. The mean percentage change in psoas area was −0.03 ± 0.05/100d in controls and −3.52 ± 0.45/100d in cirrhosis (P < .001). The mean percentage change in psoas area was −1.72 ± 0.27/100d in NAFLD, −5.28 ± 0.86/100d in ALD and −2.29 ± 0.28/100d in VH. Among cirrhotics, patients with ALD had the lowest initial muscle area and most rapid rate of reduction in muscle area. Aetiology of liver disease, model for end‐stage liver disease (MELD) and the rate of loss of muscle area were independent risk factors for survival. Conclusions Aetiology of liver disease is an independent risk factor for sarcopenia with the greatest rate of muscle loss noted in ALD. Survival in cirrhosis was dependent on initial muscle mass, rate of muscle loss and MELD score.