Trimetazidine attenuates dexamethasone-induced muscle atrophy via inhibiting NLRP3/GSDMD pathway-mediated pyroptosis

Abstract

Skeletal muscle atrophy is one of the major side effects of high dose or sustained usage of glucocorticoids. Pyroptosis is a novel form of pro-inflammatory programmed cell death that may contribute to skeletal muscle injury. Trimetazidine, a well-known anti-anginal agent, can improve skeletal muscle performance both in humans and mice. We here showed that dexamethasone-induced atrophy, as evidenced by the increase of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) expression, and the decrease of myotube diameter in C2C12 myotubes. Dexamethasone also induced pyroptosis, indicated by upregulated pyroptosis-related protein NLR family pyrin domain containing 3 (NLRP3), Caspase-1, and gasdermin-D (GSDMD). Knockdown of NLRP3 or GSDMD attenuated dexamethasone-induced myotube pyroptosis and atrophy. Trimetazidine treatment ameliorated dexamethasone-induced muscle pyroptosis and atrophy both in vivo and in vitro. Activation of NLRP3 using LPS and ATP not only increased the cleavage and activation of Caspase-1 and GSDMD, but also increased the expression levels of atrophy markers MuRF1 and Atrogin-1 in trimetazidine-treated C2C12 myotubes. Mechanically, dexamethasone inhibited the phosphorylation of PI3K/AKT/FoxO3a, which could be attenuated by trimetazidine. Conversely, co-treatment with a PI3K/AKT inhibitor, picropodophyllin, remarkably increased the expression of NLRP3 and reversed the protective effects of trimetazidine against dexamethasone-induced C2C12 myotube pyroptosis and atrophy. Taken together, our study suggests that NLRP3/GSDMD-mediated pyroptosis might be a novel mechanism for dexamethasone-induced skeletal muscle atrophy. Trimetazidine might be developed as a potential therapeutic agent for the treatment of dexamethasone-induced muscle atrophy.

Fig. 1. DEX induces muscle atrophy and pyroptosis in C2C12 myotubes.

A Cell viability of C2C12 myotubes treated with DEX (0.1, 1, 10 μM) for 24 h. n = 5 per group. B Real-time PCR analysis of expression of muscle atrophic markers (Atrogin-1 and MuRF1) in C2C12 myotubes treated with 10 μM DEX for 24 h. n = 6 per group. C Western blot analysis of expression level of Atrogin-1 and MuRF1 in C2C12 myotubes treated with 10 μM DEX for 24 h. GAPDH was used as a loading control. n = 5 per group. D Western blot analysis of expression level of NLRP3, Caspase-1, Cleaved-Caspase-1, GSDMD, and Cleaved-GSDMD in C2C12 myotubes treated with 10 μM DEX for 24 h. n = 4 per group. All experiments were performed on C2C12 mature myotubes. Data are presented as mean ± SEM. *P < 0.05 vs. Ctrl, **P < 0.01 vs. Ctrl. DEX dexamethasone, Ctrl control.

Fig. 2. Inhibition of pyroptosis alleviates DEX-induced muscle atrophy in C2C12 myotubes.

A C2C12 myotubes were transfected with negative control (siNC) or siRNA targeting GSDMD (siGSDMD). Protein levels of GSDMD were analyzed. n = 3 per group. **P < 0.01 vs. siNC. B–C C2C12 myotubes were treated with 10 μM DEX combined with siNC or siGSDMD for 24 h. B Protein levels of GSDMD, Atrogin-1, and MuRF1 were analyzed. (C) Immunofluorescence staining showed the co-localization of GSDMD (red) with MHC (green) in myotubes. The distribution of C2C12 myotubes diameter was analyzed. n = 4 per group. **P < 0.01 vs. Ctrl. ^#P < 0.05 vs. DEX + siNC, ^##P < 0.01 vs. DEX + siNC. Scale bar = 100 μm. D C2C12 myotubes were transfected with negative control (siNC) or siRNA targeting NLRP3 (siNLRP3). Protein levels of NLRP3 were analyzed. n = 3 per group. **P < 0.01 vs. siNC. (E-F) C2C12 myotubes were treated with 10 μM DEX combined with siNC or siNLRP3 for 24 h. E Protein levels of NLRP3, Caspase-1, Cleaved-Caspase-1, GSDMD, Cleaved-GSDMD, Atrogin-1, and MuRF1. F Immunofluorescence staining showed the co-localization of NLRP3 (red) with MHC (green) in myotubes. n = 4 per group. **P < 0.01 vs. Ctrl. ^#P < 0.05 vs. DEX + siNC, ^##P < 0.01 vs. DEX + siNC. Scale bar = 100 μm. NC normal control, DEX dexamethasone.

Fig. 3. TMZ alleviates DEX-induced muscle atrophy in C2C12 myotubes.

A Myotubes viability. a. C2C12 myotubes treated with TMZ (50, 100, 150, 200 μM) for 6 h. b. C2C12 myotubes treated with 10 μM DEX for 24 h and TMZ (50, 100, 150, 200 μM) in the last 6 h. n = 4–5 per group. B Real-time PCR analysis of expression of Atrogin-1 and MuRF1 in C2C12 myotubes treated with 10 μM DEX and 150 μM TMZ. n = 4 per group. C Protein levels of Atrogin-1 and MuRF1 in C2C12 myotubes treated with 10 μM DEX and 150 μM TMZ. n = 4 per group. D Protein levels of p85α PI3K, p-AKT, total AKT, p-FOXO3a, and total FOXO3a in C2C12 myotubes treated with 10 μM DEX and 150 μM TMZ. n = 4 per group. E Immunofluorescence staining for NLRP3 and GSDMD (red) in MHC (green) positive C2C12 myotubes treated with 10 μM DEX and 150 μM TMZ. The distribution of C2C12 myotubes diameter was analyzed. n = 4 per group. Scale bar; = 100 μm. **P < 0.01 vs. Ctrl. ^#P < 0.05 vs. DEX. ^##P < 0.01 vs. DEX. DEX dexamethasone; TMZ trimetazidine, Ctrl control.

Fig. 4. TMZ ameliorates DEX-induced muscle atrophy in mice.

Mice were intraperitoneally injected with 0.9% saline (Ctrl), DEX (25 mg/kg), TMZ (5 mg/kg), or DEX (25 mg/kg) +TMZ (5 mg/kg) for 10 days. A Body weight of mice. n = 8 per group. B Exercise capacity testing. a. Running distance. b. Running time. n = 8 per group. C Grip strength test. n = 8 per group. D Comparison of representative samples of dissected skeletal muscle including gastrocnemius (Gast), tibialis anterior muscle (TA), soleus muscle (Sol), and quadriceps (Quad). n = 4 per group. E Representative H&E staining of myofiber cross-section of Gast. Scale bar = 100 μm. A microscope with a 10× objective was used to capture the images. n = 4 per group. F Protein levels of Atrogin-1 and MuRF1 in Gast muscle. n = 4 per group. G Protein levels of p85α PI3K, p-AKT, total AKT, p-FOXO3a, and total FOXO3a in Gast muscle. n = 4 per group. *P < 0.05 vs. Ctrl. **P < 0.01 vs. Ctrl. ^#P < 0.05 vs. DEX. ^##P < 0.01 vs. DEX. DEX dexamethasone, TMZ trimetazidine, Ctrl control.

Fig. 5. TMZ attenuates DEX-induced pyroptosis in myotubes and mice.

A–B C2C12 myotubes were treated with 10 μM DEX for 24 h combined with or without 150 μM TMZ in the last 6 h. A mRNA levels of NLRP3, Caspase-1, and GSDMD. B Protein levels of NLRP3, Caspase-1, Cleaved-Caspase-1, GSDMD, and Cleaved-GSDMD. n = 4 per group. C Mice were intraperitoneally injected with 0.9% saline (Ctrl), DEX (25 mg/kg), TMZ (5 mg/kg), or DEX (25 mg/kg) +TMZ (5 mg/kg) for 10 days. Protein levels of NLRP3, Caspase-1, Cleaved-Caspase-1, GSDMD, Cleaved-GSDMD, IL-1β, Cleaved-IL-1β, and IL-18 were analyzed in gastrocnemius. n = 4 per group. *P < 0.05 vs. Ctrl. **P < 0.01 vs. Ctrl. ^#P < 0.05 vs. DEX. ^##P < 0.01 vs. DEX. DEX dexamethasone, TMZ trimetazidine, Ctrl control.

Fig. 6. PI3K/AKT pathway is involved in the protective effect of TMZ against DEX-induced pyroptosis and atrophy in C2C12 myotubes.

C2C12 myotubes were treated with 10 μM DEX for 24 h combined with or without 150 μM TMZ in the last 6 h. A Myotubes were further treated with 100 ng/ml LPS for 2 h and 2.5 mM ATP for 1 h. Protein levels of NLRP3, Caspase-1, Cleaved-Caspase-1, GSDMD, Cleaved-GSDMD, Atrogin-1, and MuRF1 were analyzed. n = 4 per group. B–C Myotubes were further treated with 2.5 μM PPP for 24 h. B Protein levels of p85α PI3K, p-AKT, total AKT, p-FoxO3a, and total FoxO3a. C Protein levels of NLRP3, Caspase-1, Cleaved-Caspase-1, GSDMD, Cleaved-GSDMD, Atrogin-1, and MuRF1. n = 4 per group. *P < 0.05 vs. Ctrl. **P < 0.01 vs. Ctrl. ^#P < 0.05 vs. DEX. ^##P < 0.01 vs. DEX. ^&P < 0.05 vs. DEX + TMZ. ^&&P < 0.01 vs. DEX + TMZ. DEX dexamethasone, TMZ, trimetazidine, Ctrl control, PPP Picropodophyllin.

Fig. 7. Schematic of the mechanism by which TMZ attenuates DEX-induced skeletal muscle atrophy.

TMZ possesses a protective effect against DEX-induced skeletal muscle atrophy via promoting the phosphorylation of the PI3K/AKT pathway, which in turn inhibits FoxO3a-mediated transcriptional activation of Atrogin-1 and MuRF1 and NLRP3/GSDMD pathway-mediated pyroptosis. Black arrows represent the role of DEX, red plus (+), and minus signs (−) represent the role of TMZ. DEX dexamethasone, TMZ trimetazidine.