Sphingomyelinase depresses force and calcium sensitivity of the contractile apparatus in mouse diaphragm muscle fibers

Abstract

Diseases that result in muscle weakness, e.g., heart failure, are characterized by elevated sphingomyelinase (SMase) activity. In intact muscle, SMase increases oxidants that contribute to diminished muscle force. However, the source of oxidants, specific processes of muscle contraction that are dysfunctional, and biochemical changes underlying the weakness elicited by SMase remain unknown. We tested three hypotheses: 1) SMase-induced depression of muscle force is mediated by mitochondrial reactive oxygen species (ROS), 2) SMase depresses force and calcium sensitivity of the contractile apparatus, and 3) SMase promotes oxidation and phosphorylation of myofibrillar proteins. Our experiments included intact muscle bundles, permeabilized single fibers, and isolated myofibrillar proteins. The mitochondrial-targeted antioxidant d-Arg-2',6'-dimethyl-Tyr-Lys-Phe-NH(2), decreased cytosolic oxidants and protected intact muscle bundles from weakness stimulated by SMase. SMase depressed maximal calcium-activated force by 20% in permeabilized single fibers (in kN/m(2): control 117 ± 6; SMase 93 ± 8; P < 0.05). Calcium sensitivity of permeabilized single fibers decreased from 5.98 ± 0.03 (control) to 5.91 ± 0.02 (SMase; P < 0.05). Myofibrillar protein nitrotyrosines, carbonyls, and phosphorylation were unaltered by SMase. Our study shows that the fall in specific force of intact muscle elicited by SMase is mediated by mitochondrial ROS and can be attributed largely to dysfunction of the contractile apparatus.

Fig. 1.

d-Arg-2′,6′-dimethyl-Tyr-Lys-Phe-NH₂ (SS-31) decreases cytosolic oxidant activity in sphingomyelinase (SMase)-treated muscles. Images show 2′,7′-dichlorofluorescin (DCF) fluorescence in diaphragm bundles. Original scale bars (white) = 100 μm. All images were obtained at the same magnification. Data are arbitrary units of DCF fluorescence from the entire field of view in paired hemidiaphragms treated with SMase (0.5 U/ml), SS-31/SMase (100 μM), and respective vehicles. *P < 0.05 by t-test.

Fig. 2.

SS-31 protects against SMase-induced depression of specific force (kN/m²). Force measured at 37°C, normalized for cross-sectional area; n = 5/group. Groups are control (○), SS-31/SMase (■), and SMase (•). *P < 0.05 for control or SS-31/SMase vs. SMase; #P < 0.05 for control vs. SS-31/SMase. Statistical analysis: repeated measures ANOVA and Bonferroni's post hoc test.

Fig. 3.

SMase depresses maximal specific force of myofibrillar proteins. Specific force in permeabilized single fibers. Solid and dashed lines depict line of best fit using the Hill equation. Data are from 12 (control) and 16 (SMase) permeabilized single fibers, which were averaged into 3–5 fibers/animal (n = 4). *P < 0.05.

Fig. 4.

SMase decreases calcium sensitivity of myofibrillar proteins. Left: data from Fig. 3, expressed as percentage of force at pCa 4.5 (where pCa = −log₁₀[Ca²⁺]), to illustrate decrease in calcium ion (Ca²⁺) sensitivity. Right: pCa that elicited 50% of maximal calcium-activated force (pCa₅₀). pCa₅₀ was determined from fitting the data using the Hill equation for each individual fiber. Unique symbols depict data from individual fibers (small symbols) and averaged values for each animal (large symbols). For further details, see Fig. 3. *P < 0.05.

Fig. 5.

Myofibrillar protein ·carbonyls are unchanged by SMase. Western blot images are from representative samples showing total protein gel (left pair of lanes) and probing of protein carbonyls by OxyBlot (right pair of lanes). Horizontal bar graph shows integrated intensity of protein carbonyls normalized for total protein signal/band (top: n = 3; bottom: n = 5). MBP-C, myosin-binding protein C; TnT, troponin T; Tm, tropomyosin.

Fig. 6.

Phosphorylation status of myofibrillar proteins. Left: representative samples of Western blot after probing with SimplyBlue for total protein and Pro-Q for phosphoprotein stains. SimplyBlue and Pro-Q staining of bands on ladder and phospho standard (std.) are consistent with Pro-Q probing phosphorylated proteins. TnI, troponin I; MLC, myosin light chain; TnC, troponin C. The fiber type distribution in mouse diaphragm is ∼10% type I, 30% type IIa, and 60% type IId/x (24). Thus both slow and fast isoforms of myofibrillar proteins are present in the gel, with a predominance of fast isoforms. Right: ratio of phosphorylated to total protein signal, obtained from bands on gels stained with Pro-Q and SimplyBlue, respectively. Data are expressed as percentage of mean from controls (n = 7).