The Role of Nebulin's C-terminus in Sarcomeric Structure and Function

University Of Arizona, Tucson AZ

Investigators

Abstract

? DESCRIPTION (provided by applicant): Nebulin, one of the largest known proteins, is a filamentous protein that spans almost half the length of the sarcomere, the contractile units of the muscle. Mutations in this protein can lead to a muscular disorder called nemaline myopathy, wherein patients cannot maintain normal muscle mass and contractile function, leading to difficulties in mobility and respiration. Studies in knockout mouse models coupled with large patient cohorts have led to the observation that the C-terminal domains of nebulin interacting within the Z-discs, the connective junctions between individual sarcomeres, may contribute significantly to both nebulin's normal function in healthy sarcomeres, as well as the onset of nemaline myopathy when these domains are lost. The goal of this project is to define the function of this region of nebulin and how it contributes to the formation of healthy sarcomeres. This proposal will test the hypothesis that loss of nebulin's C-terminus is sufficient for structurl changes in muscle, subsequent loss of force, and an inability for muscle to develop and undergo hypertrophy. We will study a novel mouse model that we have made which lacks the C-terminal domains of nebulin. Aim 1 is to determine the changes that occur in muscle structure and function in this model that lacks the C- terminus of nebulin. Mouse weights will be recorded throughout development and skeletal muscle weights will be compared between wild-type mice and mice homozygous for the nebulin truncation. Structural changes will be observed through transmission electron microscopy and immunofluorescence while functional changes will be quantified through the use of whole muscle mechanics. Preliminary studies have revealed an overall weight deficit in mice with truncated nebulin as well as changes in the weights of certain skeletal muscles. Aim 2 is to investigate changes protein-protein interactions with nebulin's C-terminus. The actin-binding protein CapZ, previously reported to interact with nebulin and a known regulator of actin thin filament length, will be analyzed for loss of proper localization leading to disruption of thin filament lengths. A proposed interaction between nebulin's C-terminus and the branched actin assembly protein neuronal Wiskott-Aldrich Syndrome protein (N-WASP), recently found to induce muscle hypertrophy will also be studied through the activation of this mechanism with induced growth factor-1 (IGF-1). The results of this aim will better clarify nebulin's protein function, the sarcomeric changes seen in prior knockout models, and why muscles with altered nebulin cannot undergo hypertrophy. Aim 3 is to assess the contributions of alternate muscle hypertrophy pathways. While the IGF-1 treatment may indicate an inability to undergo hypertrophy, this aim will show if muscle with truncated nebulin is able to undergo growth through both overload hypertrophy and therapeutic exercise, both of which act through pathways that could circumvent the loss of regulatory interactions in nebulin's C-terminus. The findings of this study will help elucidate nebulin's function in muscle sarcomeres and growth regulation.