Hyperammonemia induced skeletal muscle mitochondrial dysfunction results in cataplerosis and oxidative stress

Abstract

Ammonia is a cytotoxic metabolite that is removed primarily by hepatic ureagenesis in humans. Hyperammonemia occurs in advanced hepatic, cardiac, pulmonary disease, and in urea cycle enzyme deficiencies. Increased skeletal muscle ammonia uptake and metabolism are the major mechanism of non-hepatic ammonia disposal. Non-hepatic ammonia disposal occurs in the mitochondria via glutamate synthesis from α-ketoglutarate resulting in cataplerosis. We show skeletal muscle mitochondrial dysfunction during hyperammonemia in a comprehensive array of human, rodent and cellular models. ATP synthesis, oxygen consumption, generation of reactive oxygen species with oxidative stress, and tricarboxylic acid (TCA) cycle intermediates were quantified. ATP content was lower in the skeletal muscle from cirrhotic patients, hyperammonemic portacaval anastomosis rat, and C2C12 myotubes compared to appropriate controls. Hyperammonemia in C2C12 myotubes resulted in impaired intact cell respiration, reduced complex I/NADH oxidase activity and electron leak occurring at complex III of the electron transport chain. Consistently, lower NAD⁺/NADH ratio was observed during hyperammonemia with reduced TCA cycle intermediates compared to controls. Generation of reactive oxygen species resulted in increased content of skeletal muscle carbonylated proteins and thiobarbituric acid reactive substances during hyperammonemia. A cell permeable ester of α-ketoglutarate reversed the low TCA cycle intermediates and ATP content in myotubes during hyperammonemia. However, the mitochondrial antioxidant, MitoTEMPO did not reverse the lower ATP content during hyperammonemia. We provide for the first time evidence that skeletal muscle hyperammonemia results in mitochondrial dysfunction and oxidative stress. Use of anaplerotic substrates to reverse ammonia-induced mitochondrial dysfunction is a novel therapeutic approach.

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