Characterisation and strategic treatment of dystrophic muscle

Characterisation and strategic treatment of dystrophic muscle

The mdx mouse is widely used as a model for Duchenne Muscular Dystrophy, a fatal X-linked disease caused by a deficiency of the sub-sarcolemmal protein, dystrophin. This dissertation reports characterisation of the features of dystrophy in the mdx mouse, including parameters such as electrophysiological and contractile properties of dystrophic cardiac tissue, quantitative evaluation of kyphosis throughout the mdx lifespan, and contractile properties of respiratory and paraspinal muscles. Following these characterisation studies, the efficacy of antisense oligonucleotides (AOs) to induce alternative mRNA splicing in mdx skeletal muscles (diaphragm and paraspinal muscles) was evaluated. The left atria of younger (<6 weeks) and older (>15 months) mdx mice showed consistently lower basal forces and responsiveness to increased calcium, while action potential duration was significantly shorter in young mice (3 weeks) and older mice (9 and 12 months) (P<0.05). Cardiac fibrosis increased with age in mdx atria and ventricles and was elevated in young (6-8 weeks) and old (15 months) mdx compared to control mice (P<0.01). This study provided insights into DMD cardiomyopathy, and suggested that very young or old mdx mice provide the most useful models. Mdx mice show thoracolumbar kyphosis like boys with Duchenne Muscular Dystrophy. A novel radiographic index, the Kyphotic Index (KI), was developed and showed that mdx mice are significantly more kyphotic from 9 months of age, an effect maintained until 17 months (P<0.05). At 17 months, the paraspinal and respiratory muscles (latissimus dorsi, diaphragm and intercostal muscles) are significantly weaker and more fibrotic (P<0.05). Administration of AOs at four sites within the diaphragm at 4 and 5 months of age significantly increased twitch and tetanic forces compared to sham treated mdx (P<0.05). However, no difference in collagen was evident and dystrophin was not detected, possibly due to the low concentration of AO utilised. This study suggested that AOs can provide functional improvement in treated skeletal muscles. Monthly injections with AOs into the paraspinal muscles from 2 months to 18 months of age alleviated kyphosis, without significantly altering twitch and tetanic forces of latissimus dorsi, diaphragm and intercostal muscles. There was evidence of less fibrosis in diaphragm and latissimus dorsi muscles (P<0.05) and reduced central nucleation of the latissimus dorsi and intercostal muscles (P<0.05). Again, dystrophin was not detected by immunoblot. These studies indicate that very young and old mdx mice display previously uncharacterised dystrophic features, and are useful models for testing new therapies such as AOs. Low doses of AOs were shown to be safe and efficacious for long-term use, however there remains a need for testing higher concentrations and improved delivery strategies.