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Sphingomyelinase (SMase) hydrolyzes membrane sphingomyelin into ceramide, which increases oxidants in nonmuscle cells. Serum SMase activity is elevated in sepsis and heart failure, conditions where muscle oxidants are increased, maximal muscle force is diminished, and fatigue is accelerated. We tested the hypotheses that exogenous SMase and accumulation of ceramide in muscle increases oxidants in muscle cells, depresses specific force of unfatigued muscle, and accelerates the fatigue process. We also anticipated that the antioxidant N-acetylcysteine (NAC) would prevent SMase effects on muscle function. We studied the responses of C2C12 myotubes and mouse diaphragm to SMase treatment in vitro. We observed that SMase caused a 2.8-fold increase in total ceramide levels in myotubes. Exogenous ceramide and SMase elevated oxidant activity in C2C12 myotubes by 15–35% ( P < 0.05) and in diaphragm muscle fiber bundles by 58–120% ( P < 0.05). The SMase-induced increase in diaphragm oxidant activity was prevented by NAC. Exogenous ceramide depressed diaphragm force by 55% ( P < 0.05), while SMase depressed maximal force by 30% ( P < 0.05) and accelerated fatigue—effects opposed by treatment with NAC. In conclusion, our findings suggest that SMase stimulates a ceramide-oxidant signaling pathway that results in muscle weakness and fatigue.

Murine models of starvation-induced muscle atrophy demonstrate that reduced protein kinase B (AKT) function upregulates the atrophy-related gene atrogin-1/MAFbx (atrogin). The mechanism involves release of inhibition of Forkhead transcription factors, namely Foxo1 and Foxo3. Elevated atrogin mRNA also corresponds with elevated TNF in inflammatory catabolic states, including cancer and chronic heart failure. Exogenous tumor necrosis factor (TNF) increases atrogin mRNA in vivo and in vitro. We used TNF-treated C2C12 myotubes to test the hypothesis that AKT-Foxo1/3 signaling mediates TNF regulation of atrogin mRNA. Here we confirm that exposure to TNF increases atrogin mRNA (+125%). We also confirm that canonical AKT-mediated regulation of atrogin is active in C2C12 myotubes. Inhibition of phosphoinositol-3 kinase (PI3K)/AKT signaling with wortmannin reduces AKT phosphorylation (−87%) and increases atrogin mRNA (+340%). Activation with insulin-like growth factor (IGF) increases AKT phosphorylation (+126%) and reduces atrogin mRNA (−15%). Although AKT regulation is intact, our data suggest it does not mediate TNF effects on atrogin. TNF increases AKT phosphorylation (+50%) and stimulation of AKT with IGF does not prevent TNF induction of atrogin mRNA. Nor does TNF appear to signal through Foxo1/3 proteins. TNF has no effect on Foxo1/3 mRNA or Foxo1/3 nuclear localization. Instead, TNF increases nuclear Foxo4 protein (+55%). Small interfering RNA oligos targeted to two distinct regions of Foxo4 mRNA reduce the TNF-induced increase in atrogin mRNA (−34% and −32%). We conclude that TNF increases atrogin mRNA independent of AKT via Foxo4. These results suggest a mechanism by which inflammatory catabolic states may persist in the presence of adequate growth factors and nutrition.

Cachexia is common in chronic inflammatory diseases and is attributed, in part, to an elevation of circulating proinflammatory cytokines. TNF-α is the prototype in this category. IFN-γ is also thought to play a role, but the evidence supporting this model is primarily indirect. To determine the direct effects of IFN-γ stimulation on muscle cells, we selected key components of the procatabolic signaling pathways by which TNF-α stimulates protein loss. We tested two hypotheses: 1) IFN-γ mimics TNF-α signaling by increasing intracellular oxidant activity and activating MAPKs and NF-κB and 2) IFN-γ increases the expression of the ubiquitin ligases atrogin1/MAFbx and muscle-specific ring finger protein 1 (MuRF1). Results showed that treatment with IFN-γ at 60 ng/ml increased Stat1 phosphorylation after 15 min, indicating receptor activation. IFN-γ had no effect on cytosolic oxidant activity, as measured by 2′,7′-dichlorofluorescein oxidation. Nor did IFN-γ activate JNK, ERK1/2, or p38 MAPK, as assessed by Western blot. Treatment for up to 60 min did not decrease IκB-α protein levels, as measured by Western blot analysis, or the DNA binding activity of NF-κB, as measured by EMSA. After 6 h, IFN-γ decreased Akt phosphorylation and increased atrogin1/MAFbx and MuRF1 mRNA. Daily treatment for up to 72 h did not alter adult fast-type myosin heavy chain content or the total protein-to-DNA ratio. These data show that responses of myotubes to IFN-γ and TNF-α differ markedly and provide little evidence for a direct catabolic effect of IFN-γ on muscle.