POS0472 AGONISTIC STIMULATION OF GLUCAGON-LIKE PEPTIDE-1 RECEPTOR AMELIORATED INFLAMMATORY MYOPATHIES THROUGH SUPPRESSING MUSCLE FIBER NECROPTOSIS

Abstract

BackgroundWhile glucocorticoids (GC) are the cornerstone of the treatment for polymyositis (PM), GC-induced myopathy is inevitable, which further deteriorates muscle weakness. Therefore, novel therapeutic strategy that not only suppresses muscle inflammation but also improves muscle strength is awaited. We recently found that injured muscle fibers in PM undergo FASLG-mediated necroptosis1, a form of regulated cell death accompanied with release of pro-inflammatory mediators such as HMGB1, which contributes to accelerate muscle inflammation and muscle weakness. We also showed that inhibition of necroptosis or HMGB1 ameliorated the muscle weakness and muscle inflammation1 in C protein-induced myositis (CIM), a murine model of PM. Glucagon-like peptide-1 receptor (GLP-1R) agonists, which have been developed as an anti-diabetic therapy, have pleiotropic actions including anti-inflammatory effects2, suppression of muscle wasting3, and inhibition of cell death4. Accordingly, we hypothesized that GLP-1R agonists have beneficial effects on PM to recover muscle strength and to suppress muscle inflammation.ObjectivesThe aims of this study are to examine the role of GLP-1R in PM and the effect of a GLP-1R agonist on in vivo and in vitro models of PM.MethodsMuscle specimens of PM patients and CIM were examined with immunofluorescence staining for the expression of GLP-1R. The effect of PF1801, a GLP-1R agonist, on CIM was examined in monotherapy or in combination with prednisolone (PSL). As an in vitro model of PM, C2C12-derived myotubes were treated with FAS ligand (FASLG) to induce necroptosis. The levels of HMGB1, TNF-α, and IL-6 in the serum of CIM and in the culture supernatant of the in vitro model were measured by ELISA. The effect of PF1801 on the myotube necroptosis was examined using time lapse imaging 5 and its effect on the activation of AMP-activated protein kinase (AMPK), the expression of PGAM5, and ubiquitination of PGAM5 was assessed with immunoblotting. The levels of reactive oxygen species (ROS) in the myotubes were analyzed with CellROX assay. The effect of PF1801 on the expression of antioxidant molecules in the myotubes was analyzed with quantitative real-time PCR.ResultsGLP-1R was expressed on the inflamed muscle fibers of PM and CIM. The treatment with PF1801 in monotherapy or in combination with PSL suppressed CIM-induced muscle weakness and the muscle weight loss as well as the severity of histological myositis while the monotherapy with PSL did not suppress muscle weakness and muscle weight loss. PF1801 decreased the levels of inflammatory mediators such as HMGB1, TNF-α, and IL-6 in the serum of CIM. In vitro, PF1801 inhibited FASLG-induced myotube necroptosis and decreased the levels of HMGB1, TNF-α, and IL-6 in the supernatant. PF1801 activated AMPK and decreased the levels of PGAM5, which was crucial for FASLG-induced necroptosis of the myotubes. The inhibitory effect of PF1801 on myotube necroptosis was cancelled by compound C, an AMPK-kinase inhibitor, or MG132, a proteasome inhibitor, suggesting that PF1801 promoted ubiquitin-proteasome-mediated PGAM5 degradation through the activation of AMPK. Furthermore, PF1801 suppressed FASLG-induced reactive oxygen species (ROS) accumulation in myotubes, which was also crucial for the execution of necroptosis, thorough up-regulating the antioxidant molecules such as Nfe2l2, Hmox1, Gclm, and Nqo1.ConclusionGLP-1R agonist could be a novel therapy for PM that restores muscle strength as well as suppresses muscle inflammation through inhibiting muscle fiber necroptosis.