Sarcopenia in cirrhosis is caused by hyperammonemia induced skeletal muscle mitochondrial dysfunction
Samjhana Thapaliya2, Ciaran E. Fealy2, Anny Mulya2, Chris A. Flask3, John P. Kirwan2,1, Srinivasan Dasarathy1, 2
1Gastroenterology and hepatology, Cleveland Clinic Foundation, Cleveland, OH; 2PathobioIogy, Cleveland Clinic, Cleveland, OH; 3Imaging Research Core, Case Western Reserve University, Cleveland, OH
Background. Cirrhosis is a state of disordered energy metabolism that contributes to sarcopenia or loss of muscle mass. Hyperammonemia is a consistent abnormality in cirrhosis that we have previously shown as a critical mediator of sarcopenia due to impaired protein synthesis. Protein synthesis places a high energy demand on the cellular bioenergetic pathways. In the present study, we demonstrate that hyperammonemia results in abnormal skeletal muscle mitochondrial dynamics and bioenergetics. Methods. The portacaval anastamosis (PCA) rat, model of hyperammonemia and sarcopenia of liver disease and pair fed sham operated controls (n=6 each) were subjected to magnetic resonance spectroscopy to quantify in vivo, skeletal muscle ATP and phosphocreatinine (PC) content and ATP/PC ratio were determined. These data were complemented by in vitro studies in C2C12 murine myotubes exposed to 10mM hyperammonemia to study mitochondrial function. ATP production was quantified using a bioluminescence assay. Mitochondrial dynamics were evaluated by real time PCR for genes regulating mitochondrial fusion (Mfn1), & fission/mitophagy (DRP1, & DJ1). Reactive oxygen species (R〇S) was measured by a fluorometric method. Mitochondrial function was quantified using the Seahorse XF24® analyzer. Total cellular protein and ribosomal content were quantified for translational efficiency and cell size was quantified on phase contrast microscopy. Results. Gastrocnemius muscle PC content in PCA rats was 41.7±31.4% and of ATP content was 41.7±31.4% of controls rats. In C2C12 myotubes, ATP content was 20.3±3.4 μmol/mg protein lower in response to hyperammonemia compared to controls. The time course evaluation showed that this was an early abnormality beginning within 5 m and persisted to 24 h. This was accompanied by an increased generation of ROS (133.6±12.8% of controls). Mitochondrial stress test in C2C12 cells showed that hyperammonemia decreased basal respiration and increased the spare respiratory capacity at early time point. Mitochondrial dynamics were altered: specifically, relative expression of Mfn1 was 0.48±0.09, Drp1 was 8.4±2.3 fold and DJ1, 2.1 ±0.73 fold within 5 minutes and these changes persisted for to 24 h. of hyperammonemia suggesting an increase in net mitochondrial fragmentation. These changes were accompanied by reduced protein content, translational efficiency and myotubes size. Conclusions. Our observations suggest a novel hyperammonemia mediated mechanism of sarcopenia and decreased muscle protein synthesis in cirrhosis due to dysregulated mitochondrial dynamics and impaired mitochondrial function.
The following people have nothing to disclose: Samjhana Thapaliya, Ciaran E. Fealy, Anny Mulya, Chris A. Flask, John P. Kirwan, Srinivasan Dasarathy