Corticortophin releasing factor 2 receptor agonist treatment significantly slows disease progression in mdx mice

Abstract

Background: Duchenne muscular dystrophy results from mutation of the dystrophin gene, causing skeletal and cardiac muscle loss of function. The mdx mouse model of Duchenne muscular dystrophy is widely utilized to evaluate the potential of therapeutic regimens to modulate the loss of skeletal muscle function associated with dystrophin mutation. Importantly, progressive loss of diaphragm function is the most consistent striated muscle effect observed in the mdx mouse model, which is the same as in patients suffering from Duchenne muscular dystrophy.

Methods: Using the mdx mouse model, we have evaluated the effect that corticotrophin releasing factor 2 receptor (CRF2R) agonist treatment has on diaphragm function, morphology and gene expression.

Results: We have observed that treatment with the potent CRF2R-selective agonist PG-873637 prevents the progressive loss of diaphragm specific force observed during aging of mdx mice. In addition, the combination of PG-873637 with glucocorticoids not only prevents the loss of diaphragm specific force over time, but also results in recovery of specific force. Pathological analysis of CRF2R agonist-treated diaphragm muscle demonstrates that treatment reduces fibrosis, immune cell infiltration, and muscle architectural disruption. Gene expression analysis of CRF2R-treated diaphragm muscle showed multiple gene expression changes including globally decreased immune cell-related gene expression, decreased extracellular matrix gene expression, increased metabolism-related gene expression, and, surprisingly, modulation of circadian rhythm gene expression.

Conclusion: Together, these data demonstrate that CRF2R activation can prevent the progressive degeneration of diaphragm muscle associated with dystrophin gene mutation.

Figure 3

Effect of 3 months of treatment on either mdx or C57BL10 diaphragm specific force. Two-month-old mdx or C57BL10 mice were treated by daily subcutaneous injection with the indicated compound for 3 months and at the end of treatment, the diaphragms were removed and evaluated for force production. MDX Time 0, 2 month old mdx mice before treatment. MDX Vehicle, mdx mice treated for 3 months with vehicle. MDX PG-873637, mdx mice treated for 3 months with 30 ug/kg of PG-873637. C57BL10 Time 0, 2 month old C57BL10 mice before treatment. C57BL10 Vehicle, C57BL10 mice treated for 3 months with vehicle.

Figure 1

Effect of 3 months of treatment on mdx diaphragm specific force. Three-month-old mdx mice were treated by daily subcutaneous injection with the indicated compounds for 3 months and, at the end of treatment, the diaphragms were removed and evaluated for force production. Time 0, 3 month old mdx mice before treatment. Vehicle, mdx mice treated for 3 months with vehicle. Prednisone, mdx mice treated for 3 months with 1 mg/kg of prednisone. PG-873637, mdx mice treated for 3 months with 30 ug/kg of PG-873637. PG-873637 + prednisone, mdx mice treated for 3 months with the combination of 30 ug/kg PG-873637 plus 1 mg/kg prednisone.

Figure 2

Histomorphological analysis of diaphragm muscle from mdx mice treated with vehicle, prednisone, PG-87367 and PG-873637+prednisone for 3 months. Three-month-old mdx mice were treated by daily subcutaneous injection with the indicated compounds for 3 months and at the end of treatment, the diaphragms were removed and a histomorphological analysis performed. Time 0, 3 month old mdx mice before treatment. Vehicle, mdx mice treated for 3 months with vehicle. Prednisone, mdx mice treated for 3 months with 1 mg/kg of prednisone. PG-873637, mdx mice treated for 3 months with 30 ug/kg of PG-873637. PG-873637 + prednisone, mdx mice treated for 3 months with the combination of 30 ug/kg PG-873637 plus 1 mg/kg prednisone. The two photographs in each treatment group are from representative diaphragm sections from two different animals in each treatment group. All samples were hematoxylin and eosin stained.