Canine models of Duchenne muscular dystrophy and their use in therapeutic strategies

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder in which the loss of dystrophin causes progressive degeneration of skeletal and cardiac muscle. Potential therapies that carry substantial risk, such as gene- and cell-based approaches, must first be tested in animal models, notably the mdx mouse and several dystrophin-deficient breeds of dogs, including golden retriever muscular dystrophy (GRMD). Affected dogs have a more severe phenotype, in keeping with that of DMD, so may better predict disease pathogenesis and treatment efficacy. Various phenotypic tests have been developed to characterize disease progression in the GRMD model. These biomarkers range from measures of strength and joint contractures to magnetic resonance imaging. Some of these tests are routinely used in clinical veterinary practice, while others require specialized equipment and expertise. By comparing serial measurements from treated and untreated groups, one can document improvement or delayed progression of disease. Potential treatments for DMD may be broadly categorized as molecular, cellular, or pharmacologic. The GRMD model has increasingly been used to assess efficacy of a range of these therapies. A number of these studies have provided largely general proof-of-concept for the treatment under study. Others have demonstrated efficacy using the biomarkers discussed. Importantly, just as symptoms in DMD vary among patients, GRMD dogs display remarkable phenotypic variation. Though confounding statistical analysis in preclinical trials, this variation offers insight regarding the role that modifier genes play in disease pathogenesis. By correlating functional and mRNA profiling results, gene targets for therapy development can be identified.

Figure 1. Characteristic plantigrade stance in GRMD dog at 6 months of age

Pelvic limbs are shifted forward. The angle formed by the flexor surface of the tibiotarsal joint (black lines) is approximately 110° versus the 140° angle of normal standing dogs. There is hyperextension of the carpus.

Figure 2. Correlations among phenotypic tests in GRMD dogs

Scattergrams with regression lines drawn to show correlations between tibiotarsal joint angle (vertical axis in both) and body-weight-corrected tibiotarsal joint isometric tetanic extension (A) and flexion (B) force in 51 GRMD dogs at 6 months of age. Joint angle correlates strongly (p < 0.0001) with both parameters. The correlation is direct (r is positive) for extension force and inverse (r is negative) for flexion force.

Figure 3. Eccentric contraction decrement

A. Left pelvic limb of a 6-month-old GRMD dog immobilized in a stereotactic frame, with needles (covered by tape) positioned to stimulate the peroneal nerve. B. Histogram showing mean torque (N-m) generated by tetanic tibiotarsal joint flexion during a series of 10 tetanic contractions. At the conclusion of each tetanic contraction, these maximally-stimulated cranial tibial compartment muscles were forcibly stretched by the servomotor as the tibiotarsal joint was extended a further 30 degrees (see D). The mean value of the initial contractions (blue bar) was ~ 1.4 N-m, while the tenth contraction (red bar) was ~ 0.2 N-m, representing an 85% decrement. C. The characteristic tetanic mechanical potential produced by tibiotarsal joint flexion is illustrated. D. The tenth single tetanic mechanical potential of the series is followed by a sharp further deflection due to the eccentric contraction induced by the servomotor, with an immediate return to baseline.

Figure 4. GRMD cardiac studies

M-mode echocardiogram from a 4-year-old, male GRMD dog (left; Dog 2 in Table 2) showing a marked reduction in ventricular contractility at systole compared to a normal dog (right). Measurements from M-mode study for GRMD dog for Diastole/Systole: IVS 0.55 cm/0.88 cm; LV 5.75 cm/4.46 cm; LVPW 0.68 cm/0.86 cm; LV Vol 163.1 ml/90.6 ml. LV FS = 22.4%. LV EF = 44.4%.

Figure 5. GRMD MRI Studies

The four panels from left to right are MRI images from a 2-month-old GRMD carrier (A,C,E,G) and affected littermate (B,D,F,H) and 5-year-old GRMD carrier (I,K,M) and affected dog (J,L,N). A, B, I, and J are TSE-fat percentage and C, D, K, and L are TSE-fat saturation. Transverse sections of muscle have been segmented in E, F, M, and N for region-of-interest measurements and are shown in three dimensions in G and H (2-month-old dogs only). Note, particularly, the signal-intense lesions in several muscles in D and J, representing fluid accumulation, acutely, and fatty change, chronically, respectively. Signal-intense lesions seen in J reverse with fat saturation in L. Segmentation was done using ITK-SNAP (http://www.itksnap.org/pmwiki/pmwiki.php) (Yushkevich et al 2006).

Figure 6. T2 maps from GRMD and normal dogs

Comparison of the mean intensity of the cranial sartorius (A) and rectus femoris (B) muscles in T2 map of 5 GRMD (blue lines) and 10 normal littermates (red lines), at 3, 6, and 9-12 months of age. GRMD values tend to be higher at each age in both muscles but those for the cranial sartorius overlap with normal at 6 months and even more so at 9-12 months.

Figure 7. Representative sSFEMG potentials from the peroneus longus muscle from normal (A) and GRMD (B) dogs

The normal dog has minimal neuromuscular jitter and only a few action potentials at the acquisition site, while the GRMD dog has increased neuromuscular jitter and multiple action potentials.

Figure 8. MRI 50 minutes after limb perfusion of an 11-week-old GRMD dog

T2-weighted TSE MR images without (A) and with (B, C) fat saturation. Composite images have been created to orient the left and right limbs in the same plane. As a result, some proximal structures such as the colon are duplicated. Craniocaudal (top) and transverse (bottom) sections proximal to the stifle are seen in A and B. In each image, the perfused limb is on the left. Increased signal intensity is consistently seen in the perfused limb. Intensity could be either fat or fluid in A but is due to fluid alone in the fat saturation images in B and C. Intensity is greatest in the distal third of the femur and is particularly prominent in the biceps femoris muscle which is highlighted in C. In the fat saturation images in C, the perfused (red) and non-perfused (green) biceps femoris muscles were segmented for quantitative measurements. The volume and signal intensity of the biceps femoris were proportionally higher in the perfused versus non-perfused limb. Segmentation was done using ITK-SNAP (http://www.itksnap.org/pmwiki/pmwiki.php) (Yushkevich et al 2006).

Figure 9. Autologous myoblast transplantation model in normal dogs

Cells were injected percutaneously (A, right); methylene blue identifies sites of injection (A, left). Large aggregates of cells containing fluorescent microspheres are forming fascicles (circles in B and C). Muscle fibers in these fascicles stain with ATPase (pH 9.4) (B) and toluidine blue (left image in C and D) and contain fluorescent microspheres (right image in C and D) at 24 days post-transplantation.

Figure 10. Heat map of unsupervised hierarchical clustering showing differential transcript expression between GRMD and normal profiles

The heat map was prepared from 72 mRNA profiles from the cranial sartorius (CS), long digital extensor (LDE), and vastus lateralis (VL) muscles of eight GRMD and four normal dogs at 4-9 weeks and 6 months of age. Four hundred transcripts that were differentially regulated between GRMD CS profiles versus normal CS profiles (p < 0.0001; 200 transcripts) and GRMD LDE profiles versus normal LDE profiles (p < 0.0001; 200 transcripts) were clustered against the 72 mRNA profiles. Two main clusters are outlined in yellow. The top yellow cluster shows up-regulated genes (red color) for GRMD VL, CS and LDE and some normal VL profiles on the left and down-regulated transcripts (green) to the right of the white dotted lines. The bottom yellow cluster shows the opposite for all normal profiles. The profiles in between the yellow outlined clusters (VL GRMD and VL normal box) show a similar clustering pattern as the GRMD CS and normal VL profile box within the top yellow cluster. Note that there is some heterogeneity within GRMD profile expression. The heat map was prepared in HCE 3.5 Power Analysis; http://bioinformatics.cnmcresearch.org.