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 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

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 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

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

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