The Role of Nebulin and Its C-terminus in Sarcomeric Structure, Function, and Disease

Abstract

Nebulin is a large skeletal muscle protein wound around the thin filaments, with its C-terminus embedded within the Z-disk and its N-terminus extending out towards the thin filament pointed end. Studies into nebulin’s function have been limited by the conventional knockout model’s fragility and nebulin’s role in adult muscle remains poorly understood. Therefore, a nebulin knockout model that survives into adulthood is needed. Additionally, a domain-specific study of nebulin’s C-terminus would allow for a better understanding of changes at the Z-disk and how that could contribute to nemaline myopathy. This dissertation examines two novel models in order to address these points. Characterization of a conditional nebulin knockout model (Neb cKO), which delayed nebulin deletion using a MCK-Cre transgene, found severe myopathy and a persistent force deficit. Changes in MHC isoform elucidated the biological causes of these deficits. The nebulin truncation model removed the final two unique C-terminal domains, the serine-rich region and the SH3 domain (NebΔ163-165). Homozygous NebΔ163-165 mice that survive past the neonatal stage exhibit a mild weight deficit. Characterization of these mice revealed that the truncation caused a moderate myopathy phenotype reminiscent of nemaline myopathy despite the majority of nebulin being localized properly in the thin filaments. This phenotype included muscle weight loss, changes in sarcomere structure, as well as a decrease in force production. GST pulldown experiments found novel binding partners with the serine-rich region, several of which are associated with myopathies. The comparison of these two models emphasizes the role of nebulin’s C-terminus in the muscle sarcomere, finding that this small region is necessary for proper sarcomeric development and shows that its loss is sufficient to induce myopathy.