FIG 1.

Hyperammonemia impairs protein synthesis in myotubes. (A) Representative immunoblotting and densitometry of puromycin incorporation in untreated C2C12 myotubes (UnT) or those treated with 10 mM ammonium acetate (AmAc) for 6 h and 24 h. (B) Heat map of transcriptome analysis comparing untreated C2C12 myotubes to those treated with 10 mM AmAc for 3 or 24 h. (C) Heat map of C2C12 myotubes after 24 h of treatment with AmAc compared with untreated cells. (D) Ingenuity Pathway Knowledge Base (IPKB) analysis of the most enriched canonical pathways present in differentiated C2C12 myotubes after 24 h of hyperammonemia. (E) Ingenuity Pathway Analysis of the β-catenin (CTNNB1) node from the entire transcriptome (from panel B). Note that the majority of connections are downregulated upon AmAc treatment (indicated in red). (F) Cellular RNA levels from untreated myotubes or those treated with 10 mM AmAc for 6 or 24 h; from the gastrocnemius muscle of portocaval anastomosis (PCA) and sham-operated, pair-fed control (Sham) rats (n = 5 in each group); and from the skeletal muscle of patients with cirrhosis (CIR) and healthy controls (CTL) (n = 5 in each group). (G) Fold changes of the expression levels of the 45S rRNA in C2C12 myotubes treated with 10 mM AmAc for 30 min or 3, 6, or 24 h in the gastrocnemius muscle of portocaval anastomosis (PCA) and sham-operated pair-fed control rats (n = 5 each) and the skeletal muscle of patients with cirrhosis and controls (n = 5 each). rRNA expression was normalized against β-actin. (H) RNA gel of 28S and 18S bands in untreated myotubes and those treated with 10 mM AmAc for different times. (I) Effect of AmAc treatment on the polysome/monosome ratio in C2C12 myotubes. Representative polysome profiles from a sucrose gradient (20 to 47%) were obtained by taking the optical density at 254 nm (OD₂₅₄) for untreated control and 10 mM AmAc-treated C2C12 cell extracts. Positions of 80S and polysome species are indicated. A.U., arbitrary units. All cellular experiments were performed in at least 3 biological replicates, and data are expressed as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (versus the control).