The Endoplasmic Reticulum Stress And Apoptosis In Hereditary Inclusion Body Myopathy

Objective:Hereditary inclusion body myopathy is an autosomal recessive inherited neuromuscular disorder.It is caused by UDP-N-acetylglucosamine2- epimerase/acetylmannosamine kinase (GNE) gene mutation which was localized on 9P12-13. The typical symptoms of the disease are early involvement of distal muscles of low limbs, especially anterior tibial muscles, and proximal muscles are involved in late stage. The disease was slowly progressive. The characteristic pathological findings of the disease is rimmed vacuole formation, tubulofilamentous nuclear inclusions and muscle fiber atrophy with little evidence of necrotic or regenerative fibers. The distinct pathological feathers of the disease indicate its unique pathomechanism though its pathogenesis is still not known.It has been reported that inclusions contain several accumulated proteins such as Aβ, tau, synuclein and cellular prion protein. In 2004, Vattemi found ERS and UPR in s-IBM muscle and demonstrate for the first time that the ER chaperones physically associate with AβPP in s-IBM muscle, suggesting their playing a role in the s-IBM pathogenic cascade. The pathological features of s-IBM and HIBM are similar except no inflammatory cells. Up to now, no ERS about HIBM were reported. Therefore, we presume that the accumulated unfolded/misfolded proteins in HIBM muscle induce ERS and the UPR. In 2001, Yan C found that the apoptotic process in DMRV using immunohistochemical stains and in situ TUNEL methods, and ERS can trigger apoptosis by activating caspase-12. Therefore, we investigate the relationship between ERS and apoptosis using IHC and WB.Material and methods:We examined muscle specimens from 7 cases of HIBM which were obtained from open muscle biopsy in our hospital. As a control we selected 3 DMD patients' muscle, 3 PM patients' muscle and 3 normal muscle specimens confirmed by pathologic profiles. The biopsied skeletal muscle specimens were flash-frozen in isopentane chilled with liquid nitrogen, and then were made into 8μm sections to analyse the pathological features of the disease. We also made 5μm sections to detect apoptotic process in frozen muscle samples from HIBM, DMD, PM and normal control. The percentages of TUNEL positive nuclei were compared in these 4 group with in situ TUNEL methods. Then we analyse the expressions of two ER chaperones and two apoptotic proteins of HIBM patients and control using fluorescent immunohistochemistry (IHC). For the IHC, a set of antibodies against two ER chaperones—BiP/GRP78 and calnexin, and two apoptotic proteins—caspase-12 and caspase-3 were used. Double immunofluorescence used antibodies against BiP/GRP78, calnexin, caspase-12 and caspase-3 in combination with mouse monoclonal antibody against Aβ. The specimens with abnormal ER chaperones and apoptotic proteins expression showed by IHC were further analyzed with western blotting (WB) for a quantitative evaluation.Result:1. Pathological features: Rimmed vacuoles formation and muscle fiber atrophy without marked necrotic and regenerating process was the most prominent alteration under light microscope. No inflammatory cells were found. 2. TUNEL-positive myonuclei with TUNEL labeling were found predominantly in subsarcolemmal nuclei in normal-appearing muscle fibers only in HIBM. Centrally placed nuclei were occasionally positively stained. Fragmented myonuclei were also found. Neither early nor late-stage apoptotic nucleus was present in other myopathy or normal muscle control.3. In all HIBM muscle biopsies, 70 to 80% of the vacuolated muscle fibers contained, mainly in their nonvacuolated cytoplasm, numerous well-defined, various sized, plaque-like, dotty, or elongated inclusions strongly immunoreactive with the antibodies against BiP/GRP78, calnexin, caspase-12 and caspase-3. In addition, in all biopsies, 10 to 15% of the nonvacuolated, otherwise normal-appearing (on a given cross-section)fibers contained similar inclusions. By double-label fluorescence immunocytochemistry, in all abnormal muscle fibers, inclusions immunoreactive for BiP/GRP78 and caspase-3 co-localized with Aβimmunoreactivity. On serial sections pairs stained for calnexin and Aβ, or caspase-12 and Aβ, Aβimmunoreactivity was present in the same muscle fibers as those having calnexin and caspase-12 immunoreactivity.4. In HIBM muscle biopsies a 78-KD band corresponding to BiP/GRP78 was present, but not in the normal muscle. In normal and HIBM muscle biopsies calnexin migrated as a 90-KD band, caspase-12 as 50-KD and 30～40-KD bands, caspase-3 as a 35-KD band. Their expression was much stronger in HIBM than in normal control muscle biopsies.Conclusion:1. Our data disclosed that the excessive ERS and UPR are involved in HIBM.2. We proved apoptotic process in degenerating fibers of HIBM.3. The co-localization of BiP/GRP78 and Aβ, caspase-3 and Aβsuggest that accumulated unfolded/misfolded proteins activate ERS which subsequently trigger apoptosis process by activating caspase-12, and then lead to the characteristic pathological feature of HIBM—rimmed vacuoles.Significance:1. In the present project, we proved ERS and UPR in muscle fibers of HIBM with different detecting methods.2. We also proved apoptotic process in muscle fibers of HIBM with different methods. These results indicate that apoptosis may be the crucial pathomechanism of nuclear changes, rimmed vacuole formation and subsequent muscle fiber atrophy.2.The project suggest that ERS is most likely the early step, which subsequently trigger apoptosis process by activating caspase-3. The above findings may shed a light on the mechanism of muscle fiber degeneration in HIBM, and thus may offer valuable information for therapeutic exploration in future.