Bortezomib (PS-341) Treatment Decreases Inflammation and Partially Rescues the Expression of the Dystrophin-Glycoprotein Complex in GRMD Dogs

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

Immunohistochemistry of TGF-β and dystrophin in skeletal muscle.

<p><b>A and E.</b> Muscle from healthy dogs. TGF-β is detected around the vessels in muscles (A), α-dystroglycan (E) and dystrophin (I) patterns in the sarcoplasmic membrane. <b>B:</b> Muscle from CD at T1 shows greater TGF-β expression in the endomysium of the fibers and more deposition of connective tissue at T1. <b>C and D:</b> TD after treatment with bortezomib (T1) showed lower deposition of connective tissue and lower expression of TGF-β in the endomysium of the fibers. Original magnification: 40×; bar: 50 µm. <b>F, G and H:</b> Neither untreated (CD) (J) nor treated GRMD dogs (TD) (K and L) showed expression of dystrophin in the sarcoplasmic membrane, indicating that bortezomib did not rescue this protein during the treatment. Original magnification: 20×; bar: 100 µm.</p

Ultrastructural analysis of connective tissue in muscles from GRMD dogs.

<p><b>A:</b> Muscle from a healthy dog. There was a narrow endomysium space and a lower deposition of connective tissue (→). <b>B:</b> An untreated GRMD dog (CD). The endomysium of this dog exhibits a higher deposition of connective tissue and hypercontracted fibers (HF). <b>C and D:</b> Treated GRMD dogs (TD) showed a lower deposition of connective tissue and endomysium, and few fibers were hypercontracted (HF). Original magnification: 3,500×. <b>E:</b> In muscles from healthy dogs, the mitochondria were preserved and had the same electron density as the fibers, and the mitochondrial cristae were visible. <b>F:</b> GRMD dogs demonstrated abnormal mitochondria, had a higher electron density, and were smaller, and the cristae were not visible. <b>G and H:</b> Abnormal fiber (<b>NF</b>) with macrophage invasion (<b>M</b>), complete loss of membrane integrity and myofibrillar structure showing a finely granular cytoplasm. Activated fibroblasts (<b>F</b>) with a prominent rough endoplasmic reticulum (→) were present in the endomysium. Original magnification: A and B, 8,900×; C, 3,500×; D, 5,600×.</p

Serum and hematological parameters in GRMD dogs.

<p>Mean values for altered serum and hematological parameters from GRMD dogs during the nine weeks of the study. The times represent the bortezomib treatment period in the treated dogs (K1 and B7). The parameter with the greatest change was serum CK concentration, with a high magnitude range of 40 to 70 times the reference value from Kaneko et al. (1997). CK: creatine kinase</p

Immunohistochemistry and western blot for β-dystroglycan.

<p><b>A:</b> Muscle from a healthy dog shows the β-dystroglycan pattern in sarcoplasmic membranes. <b>B:</b> Untreated GRMD dog. <b>C and D</b>: Western blot and immunohistochemical analysis from a TD show higher expression of β-dystroglycan in muscle fibers than CD after treatment with bortezomib. Using ImageJ software and measuring the blot band intensity, we found that TD-K1 had a 2-fold increase in the expression of β-dystroglycan. Original magnification: 40×; bar: 50 µm.</p

Clinical signs in GRMD dogs.

<p>The main clinical signs observed in GRMD dogs during treatment with bortezomib (three, four and five months of age). There were different phenotypes, but the main clinical signs related to muscular dystrophy progression were common to all of the dogs in the study. For physiological studies of GRMD, please see the reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061367#pone.0061367-Yang1" target="_blank">[63]</a> showing an <i>in situ</i> protocol to measure the force generated by a single muscle in dogs.</p

Immunohistochemistry of phospho-NFκB in skeletal muscle.

<p><b>A and C:</b> Muscle from a CD shows higher expression of the (active) phospho-NFκB in the nuclei of muscle fibers. <b>B and D:</b> GRMD dogs (TD), after treatment with bortezomib, showed a lower expression of phospho-NFκB in the nuclei of fibers, indicating proteasome inhibition and preservation of inactive NFκB in the cytoplasm. Original magnification: A and B, 10×; bar, 200 µm; C and D, 20×; bar, 100 µm. <b>E:</b> The phospho-NFκB positive nuclei were counted in 10 random fields, with images captured at 40×. Student's <i>t</i>-test was used to evaluate these results (* p<0.05). The CD showed more phospho-NFκB-positive nuclei, indicating more activation of proteasomal activity inducing pro-apoptotic factors and inflammatory molecules.</p

Immunohistochemistry and western blot for α-dystroglycan.

<p><b>A:</b> Muscle from a healthy dog shows the α-dystroglycan pattern in sarcoplasmic membranes. <b>B:</b> Untreated GRMD dog (CD). <b>C and D:</b> Western blot and immunohistochemical analysis from a TD showing higher expression of α-dystroglycan in muscle fibers than CD after treatment with bortezomib. Using ImageJ software and measuring the blot band intensity, we suggest that TD-B7 had a 4-fold increase in the expression of α-dystroglycan. Original magnification: 40×; bar: 50 µm.</p

Histological analysis of H&E-stained skeletal muscle fibers after treatment with bortezomib and muscle collagen morphometry.

<p>A and B: CD showed a greater deposition of connective tissue in the endomysium and perimysium (▸), and the inflammatory cells formed groups or massive lesions (*) with a poor histopathological appearance. C and D: TD showed lower deposition of connective tissue in endomysium and perimysium (▸) and a lower presence of inflammatory cells (*); Original magnification: 10×; bar: 200 µm. E: Mean and SD of muscle collagen morphometry of slides stained with picrosirius red followed by quantitative analysis. Muscle from healthy dogs and TD and CD before (T0) and after (T1) treatment with bortezomib. The p-value was <0.0001 for comparing the collagen at T0 and T1 for CD. At T1 there was a statistically significant difference between the TD and CD, with higher collagen levels in CD (p = 0.0028).</p