G.P.263

Ten genes have been identified to cause nemaline myopathy when mutated and yet the mechanism by which muscle weakness occurs remains elusive. Nemaline myopathy is defined by the presence of rod-like aggregates in the muscle and, in some cases, can be accompanied by a thickening of the Z-disk, leading to the suggestion that this is their site of origin. Whilst the presence of rods defines the condition, previous studies have demonstrated there is no correlation between their frequency and the severity of disease, questioning their contribution to muscle weakness. To investigate the mechanism of disease in nemaline myopathy, and the functional consequences of rod formation, we created a zebrafish model conditionally overexpressing an EGFP tagged mutant form of skeletal muscle α -actin (ACTA1D286G-EGFP). These fish form nemaline bodies in their muscle, as evidenced by Gomori trichrome labelling and electron microscopy, and show reduced swimming activity. Through the use of in vivo imaging to examine the onset and progression of disease in vivo we identified two distinct types of ACTA1 aggregates representing successive stages in disease progression. The earlier forming rods arise from the myosepta, the site of muscle attachment in the zebrafish, with analogy to the myotendinous junction, and not the Z-disk as previously supposed. The rods are dynamic within the muscle cell, being seen to move within the cytoplasm in vivo , and are transitory in nature. Coincident with the breakdown of the rods, globular aggregation is observed, the presence of which correlates with reduced swimming performance. Our data provide new morphological markers for disease progression and suggest a novel pathogenic mechanism whereby muscle weakness results from accumulation of excess actin. Further support will be presented from additional zebrafish myopathy models and emerging data from recently identified nemaline mutations.