Modification of the myostatin and nitric oxide signalling pathways as treatments for Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease affecting 1 in 3500 live male births that arises from faulty production of the sub-sarcolemmal protein, dystrophin. Pathology associated with dystrophin deficiency is complex but primarily characterised by progressive muscular atrophy and fibrosis resulting from cycles of damage and repair. Four separate studies in this dissertation used murine models of DMD (mdx mice) to examine the effects of myostatin absence and modulation of the NO-cGMP pathway on muscle function and fibrosis. Transgenic mice lacking the ability to produce the endogenous inhibitor of muscle growth, myostatin, were generated in both dystrophic (2KO) and non-dystrophin (Mstn-/-) strains. Myostatin absence caused cardiac hypertrophy and rescued cardiac function in mdx mice but was associated with increased cardiac fibrosis. Earlier studies suggested that dietary supplementation with the nitric oxide synthase (NOS) substrate, L-arginine, improved cardiac function but also increased muscle fibrosis at higher doses. This study aimed to optimise L-arginine dosing by treating mdx mice with 1%, 2% and 5% L-arginine in drinking water and assessment of cardiac function and fibrosis. Low-dose L-arginine had minimal to no effect on both cardiac function and cardiac fibrosis. Patch-clamp, calcium imaging and cell shortening experiments showed mdx cardiomyocytes had smaller calcium transients accompanied by depressed calcium currents but retained relatively strong cell shortening. Overexpression of cardiac nNOS in mdx mice resulted in normalisation of L-type activation and inactivation as well as force-frequency relationships, increases in calcium transient height and a prolonged peak time interval. In addition, there was also evidence of an additional outward current observed in mdx cells. Chronic administration of the PDE5 inhibitor tadalafil was used to examine whether cGMP-mediated pathways affected mRNA expression of the profibrotic genes TNF-α, TGF-β, fibronectin and PC1. Tadalafil did not significantly alter expression of any of the genes tested in twelve month old mdx but the co-precipitate in manufacture, HPMCP, may possess profibrotic properties. Neither treatment altered cardiac or skeletal muscle fibrosis. Studies in this dissertation highlight potentially beneficial treatment options in addition to defining possible drawbacks. Although further research is required, controlled myostatin administration or manipulation of the NO-cGMP pathway targeted toward increasing sub-sarcolemmal nNOS show promise as palliative treatments for DMD.