Sphingomyelinase and ceramide increase reactive species and depress maximal skeletal muscle force in vitro

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Activation of Sphingomyelinase (SMase) depresses maximal muscle force: an effect blunted by antioxidants. In non‐muscle cells, SMase catalyzes the breakdown of sphingomyelin into ceramide, which increases reactive oxygen and nitrogen species (i.e., oxidants). Thus, we tested three hypotheses: i) SMase increases ceramide levels in myocytes; ii) SMase and ceramide increase oxidants in myocytes; and iii) ceramide depresses skeletal muscle force. We performed experiments in differentiated mytubes and murine diaphragm. Myotubes were exposed to SMase (0.5 U/ml), C2‐ceramide (10 μM), C6‐ceramide (10 μM), or vehicle (CTRL) for 20 min at 37°C. Diaphragm fiber bundles were exposed to C6‐ceramide in vitro (20 μM, 30 min, 37°C). Our results show that SMase increased total ceramide levels (in pmol/nmol Pi: CTRL 1.96 ± 0.09, SMase 5.47 ± 0.36; p < 0.05). Oxidant‐stimulated fluorescence was increased by C2‐ceramide (17 ± 6%; p < 0.02), C6‐ceramide (25 ± 5%; p < 0.001), and SMase (35 ± 8%; p < 0.001). Reactive nitrogen species‐dependent fluorescence was unaffected by SMase (CTRL 179.4 ± 3.6 a.u., SMase 179.5 ± 3.8 a.u.). C6‐ceramide decreased maximal diaphragm force by 56 ± 10% (p < 0.05). We conclude that SMase increases reactive oxygen species in muscle cells. This effect appears to be mediated by ceramide and results in depression of maximal skeletal muscle force.*Funding provided by NIH (R01 AR055974) and AHA‐GRA (09POST2020082)