Dexamethasone stimulates store-operated calcium entry and protein degradation in cultured L6 myotubes through a phospholipase A(2)-dependent mechanism

Abstract

Muscle wasting in various catabolic conditions is at least in part regulated by glucocorticoids. Increased calcium levels have been reported in atrophying muscle. Mechanisms regulating calcium homeostasis in muscle wasting, in particular the role of glucocorticoids, are poorly understood. Here we tested the hypothesis that glucocorticoids increase intracellular calcium concentrations in skeletal muscle and stimulate store-operated calcium entry (SOCE) and that these effects of glucocorticoids may at least in part be responsible for glucocorticoid-induced protein degradation. Treatment of cultured myotubes with dexamethasone, a frequently used in vitro model of muscle wasting, resulted in increased intracellular calcium concentrations determined by fura-2 AM fluorescence measurements. When SOCE was measured by using calcium "add-back" to muscle cells after depletion of intracellular calcium stores, results showed that SOCE was increased 15-25% by dexamethasone and that this response to dexamethasone was inhibited by the store-operated calcium channel blocker BTP2. Dexamethasone treatment stimulated the activity of calcium-independent phospholipase A(2) (iPLA(2)), and dexamethasone-induced increase in SOCE was reduced by the iPLA(2) inhibitor bromoenol lactone (BEL). In additional experiments, treatment of myotubes with the store-operated calcium channel inhibitor gadolinium ion or BEL reduced dexamethasone-induced increase in protein degradation. Taken together, the results suggest that glucocorticoids increase calcium concentrations in myocytes and stimulate iPLA(2)-dependent SOCE and that glucocorticoid-induced muscle protein degradation may at least in part be regulated by increased iPLA(2) activity, SOCE, and cellular calcium levels.

Fig. 1.

Effect of dexamethasone on cytosolic calcium concentrations in cultured L6 myotubes. L6 myotubes were cultured on collagen-coated glass coverslips and were loaded with fura-2 AM (5 μM). Myotubes were then treated with 1 μM dexamethasone (Dex; A) or observed for the same period of time in the absence of dexamethasone [control (CTR); B]. Fluorescence measurements were obtained continuously within individual myotubes for 200 min at 520 nm after 340/380-nm dual excitation. Each line represents measurements from a single myotube. C: digital images of L6 myotubes allowed to differentiate on collagen-coated glass coverslips.

Fig. 2.

Calculation of store-operated calcium entry (SOCE) in cultured L6 myotubes as area under the curve (AUC). L6 myotubes were loaded with fura-2 AM (3 μM) and suspended in calcium-free buffer. The sarcoplasmic reticulum (SR) calcium store was depleted with 1 μM thapsigargin (TG; A) or 100 nM arginine vasopressin (AVP; B), followed by the addition of calcium (1.8 mM) to the suspension buffer as indicated. Myotubes in which the SR calcium store was not depleted served as blank. Cytosolic free calcium levels ([Ca²⁺]_i) were monitored by measuring fura fluorescence at 505 nm with 340/380-nm dual-wavelength excitation. The difference in influx between TG- or AVP-treated myotubes and the blank was calculated as AUC during 100 s after addition of calcium (shaded area) and was used as a measure of SOCE.

Fig. 3.

Effect of dexamethasone on TG- and AVP-induced SOCE in L6 myotubes and myoblasts. Cultured L6 myotubes or myoblasts were treated for 24 h with 1 μM dexamethasone or corresponding concentration of solvent (0.1% ethanol; CTR). After treatment with dexamethasone for 24 h, myotubes or myoblasts were suspended and loaded with fura-2 AM (3 μM). Myotube SR calcium stores were depleted with TG (1 μM; A and B) or AVP (100 nM; C and D) in calcium-free suspension medium, followed by addition of 1.8 mM calcium. A and C depict typical tracings, and B and D represent calculations of SOCE. E and F: AVP-induced SOCE in L6 myoblasts after 24 treatment with dexamethasone (1 μM) or corresponding concentration of solvent (0.1% ethanol). SOCE was calculated as AUC as described in Fig. 2. Results in B, D, and F are means ± SE with n = 3–5 for each group. *P < 0.05 vs. control.

Fig. 4.

Effects of BTP2 and Gd³⁺ on SOCE in control and dexamethasone-treated myotubes. Cultured myotubes were treated for 24 h with 1 μM dexamethasone or corresponding concentration of solvent (0.1% ethanol). After treatment, myotubes were suspended and AVP-induced SOCE was measured in the absence or presence of 25 μM BTP2 (A) or 1 μM Gd³⁺ (B). AVP-induced SOCE was calculated as AUC during 100 s as described in Fig. 2. Results are means ± SE with n = 3–5 for each group. *P < 0.05 vs. control (no dexamethasone); ^#P < 0.05 vs. corresponding group without BTP2 or Gd³⁺. ND, not detectable.

Fig. 5.

Effects of treatment of L6 myotubes with different concentrations of dexamethasone for different periods of time on SOCE. A: cultured myotubes were treated for 24 h with different concentrations of dexamethasone, followed by measurement of AVP-dependent SOCE in suspended myotubes as AUC during 100 s after addition of 1.8 mM calcium to fura-2 AM-loaded suspended myotubes. Results are expressed as % of control (SOCE in myotubes treated for 24 h with solvent) and are given as means ± SE with n = 6 in each group. *P < 0.05 vs. 0 nM dexamethasone. B: cultured L6 myotubes were treated with 1 μM dexamethasone for different periods of time, followed by measurement of SOCE as described above. Results are means ± SE with n = 3–5 in each group. *P < 0.05 vs. CTR (no treatment with dexamethasone).

Fig. 6.

Effects of RU38486 (RU), actinomycin D (Act D), and cycloheximide (CHX) on SOCE in dexamethasone-treated L6 myotubes. Cultured myotubes were treated for 24 h with 1 μM dexamethasone or corresponding concentration of solvent (ethanol) in the absence or presence of 1 μM RU (A), 1 μg/ml Act D (B), or 10 μg/ml CHX (C). AVP-induced SOCE was determined as AUC during 100 s (AUC₁₀₀) after addition of 1.8 mM calcium to fura-2-loaded myotubes suspended in calcium-free medium as described in Fig. 2. Results are means ± SE with n = 6 in each group. *P < 0.05 vs. corresponding control group.

Fig. 7.

Effect of dexamethasone on the activity and expression of calcium-independent phospholipase A₂ (iPLA₂) in L6 myotubes. Cultured L6 myotubes were treated for 24 h with 1 μM dexamethasone or corresponding concentration (0.1%) of ethanol (CTR). A: iPLA₂ activity determined as described in experimental procedures. Results are means ± SE with n = 5 in each group. *P < 0.05 vs. control. B: iPLA₂ mRNA levels determined by real-time PCR and normalized to 18S mRNA. Results are expressed as arbitrary units, with mRNA levels in control myotubes (no dexamethasone treatment) set to 1.0. Results are means ± SE with n = 4 in each group. C: iPLA₂ protein levels determined by Western blotting performed in the absence (top) or presence (bottom) of a specific blocking peptide. Molecular masses of the bands given on right of blots were determined from a molecular weight ladder included during electrophoresis.

Fig. 8.

Effect of iPLA₂ small interfering RNA (siRNA) on iPLA₂ expression and SOCE in cultured L6 myotubes. A: cultured L6 myotubes were transfected with iPLA₂ siRNA (200 nM) or corresponding concentration of nontargeting (NT) control siRNA for 24 h. After transfection, mRNA levels for iPLA₂ were determined by real-time PCR and were normalized to 18S mRNA. Results are expressed as arbitrary units (AU), with mRNA levels in myotubes transfected with NT siRNA set to 1.0. Results are means ± SE with n = 4 for each group. *P < 0.05 vs. NT siRNA. B: L6 myotubes that had been transfected for 24 h with NT or iPLA₂ siRNA were treated for 24 h with 1 μM dexamethasone or corresponding concentration of solvent, followed by measurement of AVP-induced SOCE. SOCE was determined as described in Fig. 2 as AUC during 100 s after addition of calcium (1.8 mM) to fura-2-loaded myotubes suspended in calcium-free medium. Results are means ± SE with n = 3–5 in each group. *P < 0.05 vs. corresponding control group (no dexamethasone treatment).

Fig. 9.

Effects of BAPTA, Gd³⁺, and bromoenol lactone (BEL) on dexamethasone-induced protein degradation in cultured L6 myotubes. Protein degradation was determined as release of TCA-soluble radioactivity during 24 h in the absence or presence of 1 μM dexamethasone from proteins prelabeled with [³H]tyrosine and expressed as %/24 h. Dexamethasone treatment was performed in the absence or presence of 20 μM BAPTA (A), 100 μM Gd³⁺ (B), or 1 μM BEL (C). Results are means ± SE with n = 6 for each group. *P < 0.05 vs. corresponding control (no dexamethasone) group; ^#P < 0.05 vs. corresponding dexamethasone group.