The relationship of sphingolipid mechanisms with oxidative stress and changes in mitochondria during functional unloading of postural muscles

Abstract

Background. Prolonged inactivity of skeletal muscles is accompanied by the development of oxidative stress and changes in sphingolipid metabolism. The relationship of sphingolipid mechanisms with generation of reactive oxygen species (ROS) in muscles subjected to functional unloading has not been studied.The aim. To identify the relationship between changes in sphingomyelinase and ceramide abundance and ROS production in rat soleus muscle during functional unloading.Methods. Male Wistar rats were subjected to hindlimb suspension for 12 hours or 14 days with the acid sphingomyelinase (ASM) inhibitor amitriptyline (AMI). The levels of ASM, ceramide and ROS were determined by fluorescence microscopy on histological sections. Pro-oxidant enzymes (NADPH oxidases 2 and 4 (NOX2 and NOX4)), cytochrome c oxidase (COX IV), the regulator of mitochondrial biogenesis PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) in muscle homogenates were studied by Western blotting, which also was used for assessment of ceramide and ASM in the isolated mitochondrial fraction. The effects of sphingomyelinase and prooxidants on ceramide, ASM, ROS and NOX2 levels were studied in an ex vivo model by incubating the muscle with exogenous sphingomyelinase or H2O2.Results. 12-hour hindlimb suspension was accompanied by an increase in the level of ASM and ceramide in rat soleus muscle. Unloading for 14 days was characterized by an increase in ASM, ceramide, ROS, NOX2, NOX4 and a decrease in COX IV and PGC-1α levels. ASM and ceramide were also increased in the mitochondrial fraction of muscle. The ASM inhibitor amitriptyline partially or completely prevented the changes caused by the unloading. In the ex vivo model, the stimulating effect of exogenous sphingomyelinase on the ROS and NOX2 levels in rat soleus muscle was found, whereas H2O2 stimulated muscle ASM and ceramide production.Conclusion. A close relationship has been established between the sphingomyeli-nase pathway of ceramide formation and ROS production in skeletal muscle under conditions of functional unloading.