Sphingomyelinase (SMase) activity is elevated in inflammatory states and may contribute to muscle weakness in these conditions. Exogenous SMase depresses muscle force in an oxidant-dependent manner. However, the pathway stimulated by SMase that leads to muscle weakness is unclear. In non-muscle cells, SMase activates the Nox2 isoform of NADPH oxidase, which requires the p47phox subunit for enzyme function. We targeted p47phox genetically and pharmacologically (apocynin) to examine the role of NADPH oxidase on SMase-induced increase in oxidants and diaphragm weakness. SMase increased cytosolic oxidants (arbitrary units: control 203±15, SMase 276±22; P < 0.05) and depressed maximal force in wild type mice (N/cm2: control 20±1, SMase 16±0.6; P < 0.05). However, p47phox deficient mice were protected from increased oxidants (arbitrary units: control 217±27, SMase 224±17) and loss of force elicited by SMase (N/cm2: control 20±1, SMase 19±1). Apocynin appeared to partially prevent the decrease in force caused by SMase (n = 3 mice/group). Thus, our study suggests that NADPH oxidase plays an important role on oxidant-mediated diaphragm weakness triggered by SMase. These observations provide further evidence that NADPH oxidase modulates skeletal muscle function.

Chronic diseases (e.g., heart failure) increase sphingomyelinase (SMase) activity in the serum and skeletal muscle. SMase forms ceramide, which activates reactive oxygen species production by mitochondria and NADPH oxidase. Reactive oxygen species contribute to depression of force stimulated by SMase. p47phox is a subunit required for activation of NOX2 isoform of NADPH oxidase. We hypothesized that mice lacking p47phox would be protected from depression of force when treated with SMase. We exposed diaphragm bundles from genetic controls (C57BL/6J; n = 16) and p47phox knockout mice (C57BL/6J; n = 11) to vehicle (glycerol) or SMase (0.5 U/ml). Maximal tetanic force decreased in genetic controls (in N/cm2: Vehicle 20 ± 0.6, SMase 16 ± 0.7, P < 0.05), but was unchanged in p47phox knockout (in N/cm2: Vehicle 20 ± 1.0, SMase 19 ± 1.1). Our results suggest that SMase‐induced depression of force requires signaling through the p47phox subunit of NADPH oxidase. It appears that SMase increases reactive oxygen species production by NADPH oxidase, which leads to depression of force.Funding support: NIH 1K99HL098453‐01, 4R00HL098453‐02