Sphingomyelinase stimulates oxidant signaling to weaken skeletal muscle and promote fatigue

Abstract

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.

Fig. 1.

Schematic representation of hypotheses tested. Steps from sphingomyelin/sphingomyelinase (SMase) to oxidants are proposed on the basis of studies in nonmuscle cells (12, 19, 30, 52), while oxidants cause skeletal muscle weakness and fatigue (1, 4, 25, 37, 46, 49). Oxidants include reactive oxygen and reactive nitrogen species. NAC, N-acetylcysteine.

Fig. 2.

Increase in myotube ceramide content after SMase exposure. Left: total myotube ceramide content normalized for lipid phosphate (P_i) levels (control, n = 5; SMase, n = 3). Right: fold change in ceramide subspecies (C₁₄ to C₂₆ carbon chain length) in response to SMase exposure (0.5 U/ml). dh, Dihydroceramide. Data are means ± SE. Statistical analysis: unpaired t-test (*P < 0.05).

Fig. 3.

C2C12 myotube oxidant activity is increased by ceramide and SMase. Myotubes were exposed to vehicle (control), C₂-ceramide (C₂-Cer; 10 μM, n = 11/group), C₆-ceramide (C₆-Cer; 10 μM, n = 12/group), and SMase (0.5 U/ml, n = 12/group) for 30 min at 37°C. Bars represent percentage change in arbitrary fluorescence units [%ΔAFU; (treated − control) × 100/control]. Images are representative of DCF fluorescence in myotubes collected at ×10 magnification. Images of treated myotubes are paired with respective controls. Statistical analysis: paired t-test for arbitrary fluorescence units from treated vs. respective controls (*P < 0.05).

Fig. 4.

SMase and ceramide increase diaphragm oxidant activity. Left: representative images of DCF fluorescence in diaphragm muscle fibers (×10 magnification). Middle: arbitrary units of DCF fluorescence normalized for mean of control group. SMase effect was prevented by the antioxidant NAC. Muscles were exposed to vehicle (control, n = 4), SMase (0.5 U/ml, n = 9), or NAC/SMase (10 mM, n = 5) for 30 min at 37°C. Statistical analysis: one-way ANOVA and Bonferroni post hoc test. *P < 0.01 vs. control and NAC/SMase. Right: arbitrary units of DCF fluorescence normalized for control in diaphragm muscles treated with vehicle (control, n = 5) and C₆-ceramide (20 μM, n = 5) for 10 min; images not shown. *P < 0.05 by paired t-test.

Fig. 5.

Exogenous ceramide depresses maximal force (P_o) of diaphragm strips. Maximal force measured immediately before (0 min) and after 15 and 30 min exposure to vehicle (open bars; n = 3) and C₆-ceramide (closed bars; n = 3) at 37°C. Specific forces of control muscles at 0, 15, and 30 min were 18.1 ± 2.4, 22.9 ± 1.3, and 20.4 ± 0.2 N/cm², respectively. Statistical analysis: two-way repeated-measures ANOVA and Bonferroni post hoc test; *P < 0.05.

Fig. 6.

Time- and concentration-dependent effects of exogenous SMase on maximal force of diaphragm strips. Open circle, control; solid square, SMase. Top: force measured before (room temperature) and during exposure (37°C) to vehicle (n = 4 mice) or SMase 0.5 U/ml (n = 4 mice). Dotted line indicates onset of exposure to vehicle or SMase and change in organ bath temperature to 37°C. Bottom: force after 60 min of exposure to vehicle (control, n = 6) or SMase 0.1 (n = 3), 0.25 (n = 4), and 0.5 U/ml (n = 19). Statistical analysis: top, two-way repeated-measures ANOVA and Bonferroni post hoc test; bottom, one-way ANOVA and Dunnett's post hoc test. *P < 0.05 for control vs. SMase.

Fig. 7.

Force-frequency characteristics of diaphragm muscles. Specific forces are expressed normalized for cross-sectional area (N/cm²) or peak tetanic force (%P_o). Open circle, control (n = 6); solid square, SMase (0.5 U/ml; n = 19); shaded square, NAC/SMase (10 mM, n = 7). Solid and dotted lines are best regression fit of mean data using Hill equation. Statistical analysis: two-way repeated-measures ANOVA with Bonferroni post hoc test. *P < 0.05 for control vs. SMase; θP < 0.05 for NAC/SMase vs. control or SMase.

Fig. 8.

Diaphragm fatigue characteristics during repetitive contractions. Top: specific force during matched-frequency protocol in control (open circle, n = 6) and SMase (solid square, n = 8). Middle: specific force during matched-force protocol in SMase (71 ± 3 Hz, n = 7; solid square) and NAC/SMase (45 ± 3 Hz, n = 5; shaded square). Mean control data (top) are shown by dotted line for reference. Bottom: unstimulated force during matched-force protocol. Data from 420 to 600 s were similar to 360 s and are omitted for clarity. Statistical analysis: two-way repeated-measures ANOVA and Bonferroni post hoc test. *P < 0.05 for control vs. SMase; ϕP < 0.05 NAC/SMase vs. SMase, #P < 0.05 control vs. NAC/SMase.