P.9.11 Transgenic zebrafish expressing mutant skeletal muscle actin, acta1a, model human nemaline myopathy

Abstract

The nemaline myopathies (NMs) are a heterogeneous group of congenital myopathies defined by moderate to severe muscle weakness and accumulation of rod-like structures (nemaline bodies) in myofibers. Heterozygous dominant mutations of the skeletal muscle α-actin gene (ACTA1) account for ∼25% of all NM cases. Despite our current understanding of normal actin function, the mechanisms underlying muscle pathology remain unclear and there are no curative therapies available for patients. In order to elucidate the molecular defects behind ACTA1-related NM, we are generating a panel of transgenic zebrafish lines that express a series of disease-linked dominant mutations on the zebrafish acta1a transcript, the predominant actin expressed in fast myofibers. We initially tested whether three of these variants, acta1a H42Y, M134V and V165M would lead to a muscle phenotype by injecting the mutant mRNA into zebrafish embryos. The fish overexpressing mutant actins displayed myopathic phenotypes characterized by delayed hatching from the chorion and curved bodies to variable degrees that corresponded with the severity of disease in patients with these mutations. We next generated an acta1a V165M transgenic line that stably expresses mutant actin in all fast myofibers and characterized its neuromuscular phenotypes by morphological and histological analysis at different time points. The acta1a V165M fish display muscle weakness as evidenced by their thin bodies and curved tails at 2 days-post-fertilization (dpf) and reduced motility in touch-evoked escape response assay at 5 dpf. Whole-mount phalloidin staining at 2 dpf revealed actin aggregates in the affected fish muscle, while electron microscopy demonstrated Z-line thickening and severe myofibrillar disorganization. These results provide proof of concept that zebrafish models of actin mutations recapitulate the human disease and have robust myopathic phenotypes that may be amenable for high-throughput chemical screening.