GSDME-dependent Mitochondrial Pyroptotic Pathway In Dermatomyositis

BackgroundDermatomyositis(DM)is an acquired auto-immune muscle disease,which belongs to the idiopathic inflammatory myopathies(IIMs).Dermatomyositis is characterized by subacute onset of symmetric proximal muscle weakness,specific skin rashes and presence of pathognomonic histologic features of perifascicular atrophy(PFA)and inflammatory infiltrates in perimysial and perivascular on skeletal muscle biopsies.Different mechanisms have been proposed to explain the pathological basis of PFA in dermatomyositis,but the detailed mechanisms underlying PFA have not been incompletely clarified.Pyroptosis is a new form of programmed cell death in recent years,which is related to the generation,development and prognosis of a variety of diseases.The characteristics of pyroptosis include pore formation in the membrane,cell swelling and plasma membrane disruption,cell lysis,DNA fracture and release of a large amount of LDH.At present,GSDMD and GSDME in Gasdermin family molecules are mainly studied as pyroptotic effectors.Both of these effectors can mediate the generation of pyroptosis and have the characteristic process of pyroptosis.GSDME can be activated through the mitochondrial apoptosis pathway.In the presence of over-expression of GSDME,caspase-3 can cleave GSDME to produce N-GSDME,which can transform the caspase-dependent mitochondrial apoptosis into mitochondrial pyroptosis.perifascicular atrophy is the gold standard for the diagnosis of dermatomyositis.Significant mitochondrial dysfunction in the perifascicular atrophy of dermatomyositis have been observed in our laboratory and many literatures,such as a large number of COX negative muscle fibers and a large number of blue fibers in S/C staining.It has also been reported in the past that up-regulated protein expression in mitochondrial apoptosis pathways,such as caspase-3,has been detected in muscle biopsy specimens of children with dermatomyositis.Therefore,we hypothesized that GSDME-dependent mitochondrial pyroptosis pathway may play a role in dermatomyositis,especially in the mechanism of perifascicular atrophy.So far,there have been no study on the mechanism of pyroptosis in dermatomyositis.In this study,histochemical staining,enzyme staining,immunohistochemical staining,Western blotting,and qRT-PCR were used to fully explore the mechanism of caspase-and GSDME-dependent mitochondrial pyroptosis pathway in dermatomyositis,especially the mechanism of perifascicular atrophy.Part I Analysis of clinical features of patients with dermatomyositis and pathological changes of mitochondria at perifascicular atrophyObjective The clinical and pathological characteristics of patients with dermatomyositis were analyzed,especially the abnormal changes of mitochondrial function in the perifascicular atrophy of dermatomyositis.Method Muscle specimens were obtained retrospectively from 11 patients with definite DM with characteristic pathological findings of perifascicular atrophy on the muscle biopsies from.We summarizes all the clinical data and pathological characteristics of patients with dermatomyositis.All muscle specimens were performed by HE staining,MGT staining and SDH staining,COX staining,S/C double staining.Result In this study there were more females than males.A characteristic skin rash was present in 91%patients.18%of the patients had positive myositis specific antibody(MSA).Serum creatine kinase(CK)levels were elevated in 36%patients.Pathologically,all DM patients showed the characteristic perifascicular atrophy on muscle biopsies.Mitochondrial dysfunctions were identified in all DM patients especially in the perifascicular region.And abnormality of mitochondrial function was more evident in muscle fibers around perifascicular region than that in the center of fascicle.Some common features of perifascicular atrophy were identified in these patients,such as different degrees of perifascicular atrophy in the same muscle fascicle and much more severe atrophy in the side near the wide connective tissue gap(perimysium)than the contralateral side.In addition,a variable number of fibers with punched-out vacuoles,primarily located in the peri fascicular region,were found in all DM muscle biopsies.Conclusion mitochondrial dysfunction in the perifascicular atrophy of dermatomyositis may be the characteristic change of dermatomyositis,and may be related to the pathogenesis of dermatomyositis,especially the formation of perifascicular atrophy.Part ? GSDME-dependent mitochondrial pyroptosis in the mechanism of perifascicular atrophy in dermatomyositisObjective To observe the distribution characteristics of GSDME-dependent mitochondrial pyroptosis pathway in muscle biopsy specimens of dermatomyositis,and to study the mechanism of GSDME-dependent mitochondrial pyroptosis pathway in dermatomyositis,especially in the mechanism of perifascicular atrophy.Method Muscle specimens were obtained retrospectively from 11 patients with definite DM with characteristic pathological findings of perifascicular atrophy on the muscle biopsies.Ten cases of control patients were also retrospectively selected with overt fiber atrophy on the muscle biopsies,which consisted of six neurogenic atrophy(NA)and four selective type ? fiber atrophy(SA).Four cases were chosen as the normal control(NC).All the samples were tested by HE staining,ATPase staining,immunohistochemical staining,Western blotting,and qRT-PCR,and mitochondrial pyroptosis-associated molecules included BAK,BAX,cytochrome C,caspase-3,caspase-9,GSDME,and IL-1.Result Immunohistochemical staining results demonstrated that the expression of these markers(BAK?BAX?cytochrome C?caspase-3?caspase-9?GSDME?IL-1?)was mainly limited to the perifascicular atrophy fibers in the DM tissues.Remarkably,more prominent expression of these markers was identified in the regions closer to the wide connective tissue gap(epimysium).Compared with the all controls,the expression levels of BAK,BAX,cytochrome C,caspase-9,and caspase-3,GSDME?IL-1?in the DM muscle specimens were significantly increased(p<0.01 or p<0.001).Western blot analysis showed that BAK,BAX,cytochrome C,cleaved-caspase-9(28kDa),and cleaved-caspase-3(19kDa),GSDME-N(34kDa)?IL-1? protein levels were significantly up-regulated in the DM muscle specimens,as compared with the all controls(p<0.05 or p<0.01 or p<0.001).Moreover,the mRNA expression levels of BAK,BAX,cytochrome C,caspase-9,and caspase-3 were also up-regulated(p<0.05).Conclusion the caspase-3-and GSDME-dependent mitochondrial pyroptotic pathway are involved in the pathomechanisms of dermatomyositis,especially in perifascicular atrophy,and might be a potential treatment target for DM...