Assessment of disease activity in muscular dystrophies by noninvasive imaging

Abstract

Muscular dystrophies are a class of disorders that cause progressive muscle wasting. A major hurdle for discovering treatments for the muscular dystrophies is a lack of reliable assays to monitor disease progression in animal models. We have developed a novel mouse model to assess disease activity noninvasively in mice with muscular dystrophies. These mice express an inducible luciferase reporter gene in muscle stem cells. In dystrophic mice, muscle stem cells activate and proliferate in response to muscle degeneration, resulting in an increase in the level of luciferase expression, which can be monitored by noninvasive, bioluminescence imaging. We applied this noninvasive imaging to assess disease activity in a mouse model of the human disease limb girdle muscular dystrophy 2B (LGMD2B), caused by a mutation in the dysferlin gene. We monitored the natural history and disease progression in these dysferlin-deficient mice up to 18 months of age and were able to detect disease activity prior to the appearance of any overt disease manifestation by histopathological analyses. Disease activity was reflected by changes in luciferase activity over time, and disease burden was reflected by cumulative luciferase activity, which paralleled disease progression as determined by histopathological analysis. The ability to monitor disease activity noninvasively in mouse models of muscular dystrophy will be invaluable for the assessment of disease progression and the effectiveness of therapeutic interventions.

Figure 1. The expression of luciferase in resting and injured muscle in Pax7Cre^ER/LuSEAP mice.

(A) A luciferase-expressing cell (arrows) residing under the basal lamina (laminin staining) and expressing the satellite cell marker syndecan-4 1 week after tamoxifen administration to a Pax7Cre^ER/LuSEAP mouse. Scale bar: 10 μm. (B) Three days after an acute injury to the right TA muscle of a Pax7Cre^ER/LuSEAP mouse, luciferase signals were detectable only in the injured limb. Scale to the right of the image represents photon emission from the tissue surface and is expressed as p/s/cm²/sr (or radiance). (C) Luciferase-expressing cells contributed to regenerative myotubes and nascent myofibers during the regenerative process (days 3–10), but were absent in the uninjured muscle. Scale bar: 50 μm.

Figure 2. Monitoring disease activity in dysferlin-deficient mice by noninvasive bioluminescence imaging.

(A) Diagrammatic representation of the temporal pattern of luciferase expression in dystrophic Pax7Cre^ER/LuSEAP mice. After tamoxifen administration, a subset of satellite cells express luciferase. In response to injury or muscle degeneration, these luciferase-positive satellite cells activate and proliferate, giving rise to luciferase-positive myoblasts that ultimately differentiate and fuse to form new myofibers, which will then be luciferase positive. (B) Wild-type (left) and dysferlin-deficient (right) Pax7Cre^ER/LuSEAP mice, injected with tamoxifen at 2 months of age, imaged together at 7 months of age. Scale to the right of the image represents photon emission from the tissue surface and is expressed as p/s/cm²/sr (or radiance). (C) The luciferase signals from distal hind limb muscles of both wild-type and dysferlin-deficient mice were measured before tamoxifen administration (Pre-tam), at 3 months of age, and then monthly up to 18 months of age. P < 0.05 for each measurement between 3 and 18 months of age in the dysferlin-deficient compared with the control strain; n = 12 Dysf^+/+ compared with Dysf^–/– at each time point. (D) Luciferase signals from the distal hind limb muscles of male and female dysferlin-deficient mice (n = 12; same mice as in C).

Figure 3. Regenerative activity in hind limb muscles of Dysf^–/–/Pax7Cre^ER/LuSEAP mice as a function of age.

(A) Hind limb muscles of Dysf^–/–/Pax7Cre^ER/LuSEAP mice imaged at 3, 6, 9, and 12 months of age. The luciferase signals increase in the hind limb muscles over time, with more signals emanating from the proximal muscles after 6 months of age. Scale to the right of the image represents photon emission from the tissue surface and is expressed as p/s/cm²/sr (or radiance). (B) Luciferase signals were quantified in proximal or distal hind limb muscles of Dysf^–/–/Pax7Cre^ER/LuSEAP mice. **P < 0.05; n = 12. (C) Representative images demonstrating the accrual of luciferase-positive myofibers in quadriceps muscles of Dysf^–/–/Pax7Cre^ER/LuSEAP mice at different ages. Scale bar: 50 μm. (D) Quantitation of the percentage of luciferase-positive quadriceps fibers from mice sacrificed at 3, 6, 9, and 12 months of age. *P < 0.05; n ≥ 3; value at each time point compared with that at 3 months of age.

Figure 4. Regenerative activity in proximal and distal hind limb muscles of Dysf^–/–/Pax7Cre^ER/LuSEAP mice.

(A) Representative images of luciferase-positive fibers detected in proximal and distal hind limb muscles of 12-month-old Dysf^–/–/Pax7Cre^ER/LuSEAP mice. Scale bar: 50 μm. (B) Quadriceps muscles of dysferlin-deficient mice have increasing luciferase enzyme activity over time. *P < 0.05; n = 3. Luciferase activities were measured biochemically in muscles of mice from specific age groups, as indicated.

Figure 5. Histological analyses of muscles of Dysf^–/–/Pax7Cre^ER/LuSEAP mice over time.

(A) Cryosections of quadriceps muscles of 3-, 6-, 9-, and 12-month-old mice were stained with H&E to assess for histopathologic evidence of disease progression. (B) Quantitative analysis of the percentages of centrally nucleated myofibers in quadriceps of Dysf^–/–/Pax7Cre^ER/LuSEAP mice as a function of age. *P < 0.05, **P < 0.005; n = 3; value at each time point compared with that at 3 months of age. (C) Analysis of serial sections from muscles of mice as in panel A were stained with an antibody against eMyHc to assay for regenerating myofibers. (D) Quantitative analysis of the percentages of eMyHc-positive fibers in the quadriceps muscles of Dysf^–/–/Pax7Cre^ER/LuSEAP mice as a function of age. **P < 0.05, ***P < 0.001; n = 3; value at each time point compared with that at 3 months of age. Scale bars: 50 μm.