Bortezomib (PS-341) treatment decreases inflammation and partially rescues the expression of the dystrophin-glycoprotein complex in GRMD dogs.

Karla P C Araujo,Thais P Gaiad,Carlos E Ambrosio,Dayson F Moreira,David Feder,Maria A Miglino,Michael P Lisanti,Caio N Duarte,José E Belizario,Gloria Bonuccelli

doi:10.1371/journal.pone.0061367

Abstract

Golden retriever muscular dystrophy (GRMD) is a genetic myopathy corresponding to Duchenne muscular dystrophy (DMD) in humans. Muscle atrophy is known to be associated with degradation of the dystrophin-glycoprotein complex (DGC) via the ubiquitin-proteasome pathway. In the present study, we investigated the effect of bortezomib treatment on the muscle fibers of GRMD dogs. Five GRMD dogs were examined; two were treated (TD- Treated dogs) with the proteasome inhibitor bortezomib, and three were control dogs (CD). Dogs were treated with bortezomib using the same treatment regimen used for multiple myeloma. Pharmacodynamics were evaluated by measuring the inhibition of 20S proteasome activity in whole blood after treatment and comparing it to that in CD. We performed immunohistochemical studies on muscle biopsy specimens to evaluate the rescue of dystrophin and dystrophin-associated proteins in the muscles of GRMD dogs treated with bortezomib. Skeletal tissue from TD had lower levels of connective tissue deposition and inflammatory cell infiltration than CD as determined by histology, collagen morphometry and ultrastructural analysis. The CD showed higher expression of phospho-NFκB and TGF-β1, suggesting a more pronounced activation of anti-apoptotic factors and inflammatory molecules and greater connective tissue deposition, respectively. Immunohistochemical analysis demonstrated that dystrophin was not present in the sarcoplasmic membrane of either group. However, bortezomib-TD showed higher expression of α- and β-dystroglycan, indicating an improved disease histopathology phenotype. Significant inhibition of 20S proteasome activity was observed 1 hour after bortezomib administration in the last cycle when the dose was higher. Proteasome inhibitors may thus improve the appearance of GRMD muscle fibers, lessen connective tissue deposition and reduce the infiltration of inflammatory cells. In addition, proteasome inhibitors may rescue some dystrophin-associated proteins in the muscle fiber membrane.

Highlights

Golden retriever muscular dystrophy (GRMD) is a degenerative myopathy corresponding to Duchenne muscular dystrophy (DMD) in humans
Histology and ultrastructural analysis At the first (T0) biopsy, which was made at three months of age and before bortezomib therapy, the muscle fibers showed myofiber hypertrophy, fiber necrosis and regeneration
Bortezomib was able to reduce the infiltration of inflammatory cells into the muscle fibers of treated dogs (TD), and the dogs had a lower deposition of connective tissue as a result of necrosis and muscle atrophy than control dogs (CD)

Summary

Introduction

Golden retriever muscular dystrophy (GRMD) is a degenerative myopathy corresponding to Duchenne muscular dystrophy (DMD) in humans. Both GRMD and DMD are caused by the absence of a functional dystrophin protein. As GRMD dogs closely resemble DMD patients, both in terms of body weight and in the pathological expression of the disease [7], they are excellent animal models for the study of pathogenic mechanisms and therapeutic interventions. The loss of dystrophin leads to the absence of or a great reduction in the components of the DGC, as has been described for skeletal muscle fibers from DMD patients and mdx mice [9], [10]

Methods

Results

Discussion

Conclusion