Myricanol rescues dexamethasone-induced muscle dysfunction via a sirtuin 1-dependent mechanism

Abstract

Background: Muscle atrophy and weakness are adverse effects of high dose or the sustained usage of glucocorticoids. Loss of mitochondria and degradation of protein are highly correlated with muscle dysfunction. The deacetylase sirtuin 1 (SIRT1) plays a vital role in muscle remodelling. The current study was designed to identify myricanol as a SIRT1 activator, which could protect skeletal muscle against dexamethasone-induced wasting.

Methods: The dexamethasone-induced atrophy in C2C12 myotubes was evaluated by expression of myosin heavy chain, muscle atrophy F-box (atrogin-1), and muscle ring finger 1 (MuRF1), using western blots. The mitochondrial content and oxygen consumption were assessed by MitoTracker staining and extracellular flux analysis, respectively. Muscle dysfunction was established in male C57BL/6 mice (8-10 weeks old, n = 6) treated with a relatively high dose of dexamethasone (25 mg/kg body weight, i.p., 10 days). Body weight, grip strength, forced swimming capacity, muscle weight, and muscle histology were assessed. The expression of proteolysis-related, autophagy-related, apoptosis-related, and mitochondria-related proteins was analysed by western blots or immunoprecipitation.

Results: Myricanol (10 μM) was found to rescue dexamethasone-induced muscle atrophy and dysfunction in C2C12 myotubes, indicated by increased expression of myosin heavy chain (0.33 ± 0.14 vs. 0.89 ± 0.21, *P < 0.05), decreased expression of atrogin-1 (2.31 ± 0.67 vs. 1.53 ± 0.25, *P < 0.05) and MuRF1 (1.55 ± 0.08 vs. 0.99 ± 0.12, **P < 0.01), and elevated ATP production (3.83 ± 0.46 vs. 5.84 ± 0.79 nM/mg protein, **P < 0.01), mitochondrial content (68.12 ± 10.07% vs. 116.38 ± 5.12%, *P < 0.05), and mitochondrial oxygen consumption (166.59 ± 22.89 vs. 223.77 ± 22.59 pmol/min, **P < 0.01). Myricanol directly binds and activates SIRT1, with binding energy of -5.87 kcal/mol. Through activating SIRT1 deacetylation, myricanol inhibits forkhead box O 3a transcriptional activity to reduce protein degradation, induces autophagy to enhance degraded protein clearance, and increases peroxisome proliferator-activated receptor γ coactivator-1α activity to promote mitochondrial biogenesis. In dexamethasone-induced muscle wasting C57BL/6 mice, 5 mg/kg myricanol treatment reduces the loss of muscle mass; the percentages of quadriceps and gastrocnemius muscle in myricanol-treated mice are 1.36 ± 0.02% and 0.87 ± 0.08%, respectively (cf. 1.18 ± 0.06% and 0.78 ± 0.05% in dexamethasone-treated mice, respectively). Myricanol also rescues dexamethasone-induced muscle weakness, indicated by improved grip strength (70.90 ± 4.59 vs. 120.58 ± 7.93 g, **P < 0.01) and prolonged swimming exhaustive time (48.80 ± 11.43 vs. 83.75 ± 15.19 s, **P < 0.01). Myricanol prevents dexamethasone-induced muscle atrophy and weakness by activating SIRT1, to reduce muscle protein degradation, enhance autophagy, and promote mitochondrial biogenesis and function in mice.

Conclusions: Myricanol ameliorates dexamethasone-induced skeletal muscle wasting by activating SIRT1, which might be developed as a therapeutic agent for treatment of muscle atrophy and weakness.

Keywords: Autophagy; Dexamethasone; Muscle atrophy; Myricanol; PGC-1α; SIRT1.

Figure 1

MY rescues DEX‐induced muscle atrophy in C2C12 myotubes. (A) Chemical structure of MY. (B) Cell viability of C2C12 myotubes treated with DEX and different concentrations of MY. (C) Expression levels of MyHC, atrogin‐1, and MuRF1. GAPDH was used as a loading control. Data are shown as mean ± SD, n = 6–9. *P < 0.05, **P < 0.01, MY vs. DEX. ^# P < 0.05, ^## P < 0.01, control vs. DEX. DEX, dexamethasone; MuRF1, muscle ring finger 1; MY, myricanol; MyHC, myosin heavy chain.

Figure 2

MY protects against DEX‐induced muscle atrophy through activating SIRT1. (A) MY increased SIRT1 expression dose‐dependently in DEX‐treated C2C12 myotubes. (B) Interactive sites between MY and SIRT1 by docking analysis. (C) MY enhanced SIRT1 deacetylase activity in C2C12 myotubes. (D) EX‐527 almost reversed MY‐induced changes of SIRT1, MuRF1, and atrogin‐1 expression in DEX‐treated myotubes. (E) MY increased phosphorylation of Akt and FoxO3a in DEX‐treated C2C12 myotubes. GAPDH was used as a loading control. Data are shown as mean ± SD, n = 6–9. *P < 0.05, **P < 0.01, MY or EX‐527 vs. DEX. ^## P < 0.01, control vs. DEX. DEX, dexamethasone; MY, myricanol; SIRT1, sirtuin 1.

Figure 3

MY reverses DEX‐induced loss of mitochondrial content and function in C2C12 myotubes. (A) MY increased mitochondrial content in DEX‐treated C2C12 myotubes, as assessed by MitoTracker Green staining. Scale bar = 100 μm. The images were captured using a Leica TCS SP8 Confocal Laser Scanning Microscope System with a 10× objective. (B) The effect of MY on ATP concentration in DEX‐treated C2C12 myotubes. (C) MY increased the expression levels of Cox2, Tom20, UCP3, and PGC‐1α. GAPDH was used as a loading control. (D) MY reversed DEX‐induced decrease of PGC‐1α expression and increase of acetylated PGC‐1α level, which were abolished by co‐treatment of EX‐527. (E) MY reversed DEX‐induced increases of total and acetylated FoxO3a levels, which were abolished by co‐treatment of EX‐527. (F) MY increased OCR in DEX‐treated C2C12 myotubes, as assessed by Seahorse assay. Data are shown as mean ± SD, n = 6–9. *P < 0.05, **P < 0.01, MY vs. DEX. ^# P < 0.05, ^## P < 0.01, control vs. DEX or EX‐527. DEX, dexamethasone; MY, myricanol; OCR, oxygen consumption rate; UCP3, uncoupling protein 3.

Figure 4

Effects of MY on apoptosis and autophagy in DEX‐treated C2C12 myotubes. (A) MY elevated the expression of autophagy‐related proteins LC3 and Beclin1 and suppressed the expression of p62. (B) MY increased lysosome content in DEX‐treated C2C12 myotubes, as assessed by LysoTracker fluorescence staining. Scale bar = 100 μm. The images were captured using a Leica TCS SP8 Confocal Laser Scanning Microscope System with a 10× objective. (C) Expression of apoptosis‐associated proteins, Bcl‐2, Bax, cleaved caspase‐3. GAPDH was used as a loading control. (D) Co‐treatment of EX‐527 abolished the effect of MY on expression of apoptosis‐associated proteins. Data are shown as mean ± SD, n = 6–9. *P < 0.05, **P < 0.01, MY or EX‐527 vs. DEX. ^## P < 0.01, control vs. DEX. DEX, dexamethasone; MY, myricanol.

Figure 5

MY protects against DEX‐induced muscle dysfunction in mice. (A) Body weight. [two‐way ANOVA: time effect F _{(10, 180)} = 60.98, P < 0.0001; treatment effect F _{(6, 18)} = 11.96, P < 0.0001; interaction F _{(60, 180)} = 11.26, P < 0.0001]. (B) Grip strength. [two‐way ANOVA: time effect F _{(5, 140)} = 81.00, P < 0.0001; treatment effect F _{(7, 28)} = 152.00, P < 0.0001; interaction F _{(35, 140)} = 6.40, P < 0.0001]. (C) Forced swimming time. (D) Comparison of representative samples of dissected skeletal muscle, including Gast, soleus, EDL, Quad, and TA. (E) The ratios of Gast and Quad muscle weight to body weight. (F) Representative H&E staining of myofiber cross section of Gast. Scale bar = 100 or 50 μm on top and bottom, respectively. A microscope with a 10× or 20× objective was used to capture the images. (G) The cross‐sectional diameter of Gast muscle fibre. Ctrl: PEG 400 solution; ctrl + MY‐50: PEG 400 solution with 50 mg/kg MY; DEX: PEG 400 solution with 25 mg/kg dexamethasone; DEX + MY‐5: DEX solution with 5 mg/kg MY; DEX + MY‐50: DEX solution with 50 mg/kg MY; DEX + EX‐527: DEX solution with 10 mg/kg EX‐527; DEX + MY‐50 + EX‐527: DEX solution with 50 mg/kg MY and 10 mg/kg EX‐527. Data are shown as mean ± SD, n = 6. *P < 0.05, **P < 0.01, DEX + MY‐5 vs. DEX. & P < 0.05, && P < 0.01, DEX + MY‐50 vs. DEX. ^# P < 0.05, ^## P < 0.01, control vs. DEX. ANOVA, analysis of variance; DEX, dexamethasone; EDL, extensor digitorum longus; Gast, gastrocnemius; MY, myricanol; Quad, quadriceps; TA, tibialis anterior.

Figure 6

Western blotting analysis of key proteins in Gast muscle. (A) The expression of SIRT1, atrogin‐1, MuRF1, phosphorylated Akt, total Akt, phosphorylated FoxO3a, and total FoxO3a. (B) The expression of LC3, Beclin1, p62, cleaved caspase‐3, Bcl‐2, and Bax. (C) The expression of PGC‐1α, Tom20, UCP3, and Cox2. β‐actin was used as a loading control. Data are shown as mean ± SD, n = 6. *P < 0.05, **P < 0.01, DEX + MY‐5 vs. DEX. & P < 0.05, && P < 0.01, DEX + MY‐50 vs. DEX. ^# P < 0.05, ^## P < 0.01, control vs. DEX. DEX, dexamethasone; MuRF1, muscle ring finger 1; MY, myricanol; SIRT1, sirtuin 1; UCP3, uncoupling protein 3.

Figure 7

Schematic models of molecular targets of MY in DEX‐induced muscle atrophy. DEX, dexamethasone; MY, myricanol.