Sphingomyelinase promotes oxidant production and skeletal muscle contractile dysfunction through activation of NADPH oxidase

Abstract

Elevated concentrations of sphingomyelinase (SMase) have been detected in a variety of diseases. SMase has been shown to increase muscle derived oxidants and decrease skeletal muscle force; however, the sub-cellular site of oxidant production has not been elucidated. Using redox sensitive biosensors targeted to the mitochondria and NADPH oxidase (Nox2), we demonstrate that SMase increased Nox2-dependent ROS and had no effect on mitochondrial ROS in isolated FDB fibers. Pharmacological inhibition and genetic knockdown of Nox2 activity prevented SMase induced ROS production and provided protection against decreased force production in the diaphragm. In contrast, genetic overexpression of superoxide dismutase within the mitochondria did not prevent increased ROS production and offered no protection against decreased diaphragm function in response to SMase. Our study shows that SMase induced ROS production occurs in specific sub-cellular regions of skeletal muscle; however, the increased ROS does not completely account for the decrease in muscle function.

Keywords: Nox2; ROS; force; skeletal muscle; sphingomyelinase.

Figure 1

SMase increases ROS production through Nox2 in FDB fibers. SMase increased DCF fluorescence in WT FDB fibers (black bars), which could be completely inhibited upon pre-incubation with gp91-ds (gray bars). There was no time-dependent change in the absence of SMase (white bars). p ≤ 0.05 ^*significantly different from vehicle treated control, ^#significantly different from baseline in at least n_animals = 3, n_cells = 31.

Figure 2

SMase increased Nox2 ROS production in WT FDB fibers with no effect on mitochondrial ROS. (A,B) Representative images of SMase induced changes in p47-roGFP and mito-roGFP fluorescence, respectively. (C) Nox2 ROS (p47-roGFP) in FDBs from WT animals was elevated following SMase at both 30 and 60 min. H₂O₂ and DTT (100 μM and 10 mM, black bars) resulted in further oxidation and reduction of p47-roGFP. (D) Mitochondrial ROS (mito-roGFP) did not change in response to SMase. H₂O₂ and DTT (100 μM and 10 mM, black bars) resulted in further oxidation and reduction of mito-roGFP. Fluorescence values were normalized to non-drug treated baseline measurements. p ≤ 0.05 ^*significantly different from baseline in at least n_animals = 4, n_cells = 11.

Figure 3

Genetic deletion of Nox2 prevents SMase induced ROS. (A) Nox2 ROS (p47-roGFP) was not altered with SMase in FDBs from Nox2^−/y mice. (B) Mitochondrial ROS (mito-roGFP) did not change in response to SMase. H₂O₂ and DTT (100 μM and 10 mM, black bars) resulted in further oxidation and reduction of both p47-roGFP and mito-roGFP. Fluorescence values were normalized to non-drug treated baseline measurements in at least n_animals = 4, n_cells = 12.

Figure 4

SMase increased Nox2 ROS production in FDB fibers from MnSOD overexpressing mice. (A) Representative DNA gel verifying MnSOD transgene overexpression. (B) SMase increased Nox2 ROS (p47-roGFP) in muscle from MnSOD overexpressing animals at both 30 and 60 min. (C) Mitochondrial ROS (mito-roGFP) did not change in response to SMase. H₂O₂ and DTT (100 μM and 10 mM, black bars) resulted in further oxidation and reduction of both p47-roGFP and mito-roGFP. Fluorescence values were normalized to non-drug treated baseline measurements. p ≤ 0.05 ^*significantly different from baseline in at least n_animals = 3, n_cells = 10.

Figure 5

Genetic deletion of Nox2 protected against diaphragm force decrement over time. Peak tetanic force was decreased at both 30 and 60 min following SMase administration compared with respective controls. (A) Genetic deletion of Nox2 protected against SMase induced force loss compared with WT. Black solid = WT control, Black dash = WT SMase, Red solid = Nox2^−/y control, Red dash = Nox2^−/y SMase. (B) Overexpressing MnSOD did not affect force decrement compared to WT. Black solid = WT control, Black dash = WT SMase, Red solid = MnSOD control, Red dash = MnSOD SMase. Force values at 30 and 60 min were normalized to force measured in the absence of SMase (baseline). p ≤ 0.05 ^*All genotypes significantly different from respective control, ^#Nox2^−/y SMase significantly different from WT SMase in at least n_animals = 6.

Figure 6

Nox2 deletion protected against SMase induced decreased force. Force was decreased at all frequencies following SMase administration compared with respective controls. (A) SMase induced force loss was prevented in diaphragm muscle from Nox2^−/y mice compared to WT at all frequencies. Nox2^−/y controls produced greater force at higher frequencies compared with WT control. Black solid = WT control, Black dash = WT SMase, Red solid = Nox2^−/y control, Red dash = Nox2^−/y SMase. (B) MnSOD overexpression did not prevent force loss at any frequency compared to WT SMase. Black solid = WT control, Black dash = WT SMase, Red solid = MnSOD control, Red dash = MnSOD SMase. P ≤ 0.05 ^*All genotypes significantly different from respective control, ^#Nox2^−/y SMase significantly different from WT SMase, ^**Nox2 control significantly different from WT control in at least n_animals = 4.

Figure 7

Genetic ablation of Nox2 or MnSOD overexpression does not protect against SMase induced fatigue. All genotypes fatigued approximately 75–80%. SMase induced an increased rate of fatigue in diaphragm from WT (A,B, black dashed), Nox2^−/y (A, red dashed), and MnSOD (B, red dashed) compared with their respective controls (solid lines). All force values were normalized to initial force. p ≤ 0.05 ^*All genotypes significantly different from respective control, ^†Nox2^−/y and MnSOD significantly different from respective controls in at least n_animals = 6.