Year of Publication
Doctor of Philosophy (PhD)
Dr. Michael Reid
In many chronic inflammatory diseases, patients suffer from skeletal muscle weakness, exacerbating their symptoms. Serum levels of tumor necrosis factor-alpha (TNF) and sphingomyelinase are increased, suggesting their possible role in the progression of this weakness. This dissertation focuses on the role that reactive oxygen species (ROS) and nitric oxide (NO) play in mediating TNF-induced skeletal muscle weakness and to what extent sphingolipid signaling mediates cellular response to TNF.
The first aim of this work was to identify which endogenous oxidant species stimulated by TNF contributes to skeletal muscle weakness. In C57BL/6 mice (n=38), intraperitoneal injection of TNF elicited a 25% depression of diaphragm contractile function. In separate experiments, diaphragm fiber bundles harvested from mice (n=39) and treated with TNF ex vivo showed a 38% depression of contractile function compared to untreated controls. Using ROS and NO-sensitive fluorescence microscopy in parallel with a genetic knockout animal model, TNF-induced contractile dysfunction was found to be mediated by NO generated by a specific isoform of nitric oxide synthase (NOS), nNOS. Basal levels of ROS were necessary co-mediators, but were not sufficient to elicit TNF-induced diaphragm weakness.
The second aim of this dissertation was to investigate the extent to which sphingolipids could serve as a signaling cascade post-TNF stimulus leading to the generation of NO in skeletal muscle. The effects of TNF exposure in C2C12 skeletal muscle cells were studied in vitro using mass spectroscopy to measure sphingolipid metabolism and fluorescent microscopy to quantify oxidant production. TNF exposure was associated with significant mean increases in sphingosine (+52%), general oxidant activity (+33%), and NO production (+14%). These increases were due to specific modulation of nNOS as demonstrated by siRNA knockdown of neutral ceramidase and nNOS, and confirmed by pharmacologic inhibition using N-Oleoylethanolamine and di-methylsphingosine.
In summary, these findings confirm NO as a major causative oxidant contributing to TNF’s deleterious phenotype in skeletal muscle. Moreover, the work suggests a new role for sphingosine in skeletal muscle and warrants further study of the enzymatic regulation of sphingosine to advance the discovery of new therapies for patients suffering from chronic inflammation.
Stasko, Shawn, "Defining the Role of Reactive Oxygen Species, Nitric Oxide, and Sphingolipid Signaling in Tumor Necrosis Factor - Induced Skeletal Muscle Weakness" (2013). Theses and Dissertations--Physiology. 12.