Pathophysiological Roles Of TWEAK/Fn14 And MuRF-1 In Immune-Mediated Necrotizing Myopathy

Objective: To investigate the expression of Tumor necrosis factor(TNF)-like weak inducer of apoptosis(TWEAK),fibroblast growth factor-inducible 14(Fn14)and Muscle RING finger-1(MuRF-1)in muscles biopsies from patitens with immune-mediated necrotizing myopathy(IMNM)and explore the underlying mechanisms.Methods: The expression of TWEAK,Fn14,MuRF-1 and related molecules were detected using immunostaining and quantitative real-time PCR.Then,human myoblasts were treated with recombinant TWEAK protein in vitro to explore the mechanisms of TWEAK/Fn14 involved in muscles injury.Serum levels of TWEAK and Fn14 were detected by enzyme-linked immuno sorbent assay,and the correlation between them and disease severity was analyzed.Finally,human myoblasts were transfected with small interfering RNA(siRNA)to inhibit the expression of MuRF-1 and explore the roles of MuRF-1 on muscle fiber regeneration.Results:1.TWEAK and Fn14 were overexpressed in IMNM muscles,which may be involved in several pathophysiological processes,including myofibers necrosis,regeneration and inflammatory infiltration.Human myoblasts viability was significantly decreased upon TWEAK treatment,and the expression of Fn14,chemokines and cytokines were significantly upregulated.Increased serum Fn14 levels positively correlated with muscle weakness,suggesting Fn14 as a promising biomarker for evaluating disease severity.2.The expression of MuRF-1 was upregulated in IMNM muscles,and MuRF-1 staining predominantly presented in regenerating myofibers.Inhibition of MuRF-1 expression repressed human myoblast proliferation and differentiation,leading to reduced myofibers formation.Conclusions: TWEAK and Fn14 were overexpressed in IMNM muscles,which may play roles in skeletal muscle damage and repair.Serum Fn14 levels may be a promising biomarker for evaluating disease severity.MuRF-1 may contribute to IMNM muscles injury repair in by regulating myofibers regeneration.Part Ⅰ:TWEAK/Fn14 participate in muscular pathogenesis in immune-mediated necrotizing myopathy Objective: TWEAK and its receptor fibroblast growth factor-inducible 14(Fn14),are involved in various inflammatory conditions.This study aimed to investigate the expression of TWEAK and Fn14 in IMNM muscle and explore the underlying mechanisms.Methods: Muscle biopsies from patients with IMNM(n=37)and controls(n=11)were collected.The expression of TWEAK,Fn14 and related downstream cytokines,as well as chemokines were detected using immunostaining and quantitative real-time PCR.Human myoblasts were treated with recombinant TWEAK protein in vitro.Then cell viability and TWEAK/Fn14 downstream cytokines and chemokines were evaluated.Serum samples from 25 IMNM patients and 18 healthy controls were collected.Serum levels of TWEAK and Fn14 were detected by enzyme-linked immuno sorbent assay,and the correlation between them and disease severity was analyzed.Results:1.TWEAK and Fn14 were overexpressed in IMNM muscle biopsies.TWEAK staining tended to be distributed in the endomysium,where inflammatory infiltrates were always present.Positive Fn14 staining was observed on the sarcolemma and sarcoplasm of some myofibers in IMNM biopsies,while physiological Fn14 staining was noted on capillary endothelial cells.2.The percentage of Fn14 positive myofibers positively correlated with disease severity,myonecrosis,regeneration and inflammation infiltrates.Fn14 positive myofibers tended to be surrounded or invaded by CD68+ macrophages.3.The mRNA expression of TWEAK/Fn14 downstream chemokines,cytokines and other related molecules were also significantly upregulated in IMNM biopsies.4.Human myoblasts viability was significantly decreased upon TWEAK treatment,and the expression of Fn14,chemokines and cytokines were significantly upregulated.5.Serum TWEAK levels tended to be decreased in patients with IMNM compared to healthy controls,but there was no significant difference.6.Serum Fn14 levels were increased in patients with IMNM than in healthy controls,and positively correlated with muscle weakness.Conclusions: TWEAK and Fn14 were overexpressed in IMNM muscles,which may be involved in several pathophysiological processes,including myofibers necrosis,regeneration and inflammatory infiltration.Increased serum Fn14 levels positively correlated with muscle weakness,suggesting Fn14 as a promising biomarker for evaluating disease severity.Part Ⅱ:The role of MuRF-1 in immune-mediated necrotizing myopathy Objective: MuRF-1 plays a key role in the degradation of skeletal muscle proteins and has been known as a muscle atrophy-related marker.However,how MuRF-1 involved in IMNM is still unknown.Here we sought to investigate the expression of MuRF-1 and explore its role in IMNM.Methods: Muscle biopsies from patients with IMNM(n=37)were analyzed and compared to biopsies from patients with dermatomyositis(DM,n=13),dysferlinopathy(n=9)and controls(n=7)using immunostaining.Human myoblasts were transfected with MuRF-1siRNA to inhibit the expression of MuRF-1 in vitro study.The effect of MuRF-1 on myogenesis was detected by quantitative real-time PCR,western bolt,and immunostaining.Results:1.MuRF-1 staining could be observed in IMNM,DM and dysferlinopathy biopsies,but was more abundant in IMNM and DM.The percentage of MuRF-1 positive myofibers was significantly higher in IMNM than in dysferlinopathy.2.MuRF-1 staining predominantly presented in regenerating myofibers but not in atrophic myofibers.3.MuRF-1-positive fibers tended to be distributed around necrotic myofibers and damaged myofibers,suggesting the involvement of MuRF-1 in IMNM muscle injury repair.4.MuRF-1 expression was upregulated during the process of skeletal myogenesis in vitro study.5.Inhibition of MuRF-1 expression repressed human myoblast proliferation and differentiation,leading to reduced myofibers formation.6.MuRF-1 may be involved in myoblast proliferation by regulating the expression of Cyclin A.7.MuRF-1 may be involved in myoblast differentiation by regulating the expression of myogenin.Conclusions: MuRF-1 is required for skeletal muscle regeneration by regulating myoblast proliferation and differentiation,contributing to muscle injury repair in IMNM.MuRF-1immunostaining may be a useful tool to help clinicians differentiate dysferlinopathy from IMNM.