Nebulin is an important but poorly understood sarcomeric protein found in skeletal muscle. Our previous studies with nemaline myopathy patients deficient in nebulin (NEM2 patients) revealed that nebulin protein loss results in thin filaments of reduced length, supporting that nebulin is critical for regulation of thin filament length (TFL). Reduced TFL in NEM2 patients is likely part of their severe muscle weakness because it is expected to reduce force on the descending limb of the force-sarcomere length relationship. How nebulin contributes to TFL control is controversial. To critically test nebulin’s role in TF length specification we made two novel mouse models, one in which nebulin is shortened by deleting super-repeats (SRs) 9-11 and another in which nebulin is lengthened by duplicating the same SRs. In Aim 1 we will used these contrasting and powerful models to study the role of nebulin in TFL regulation and, importantly, the functional consequence of TFL mis-specification. These studies are clinically important for multiple muscle diseases where mis-specification of TFL occurs. Nebulin is also likely to contribute to sarcomeric organization through its position in the Z-disk, where interactions with local proteins might contribute to Z-disk width regulation, myofibrillar alignment and hypertrophy signaling. It has been reported that nebulin’s C-terminal interaction with the actin assembly protein neuronal Wiskott-Aldrich syndrome protein (N-WASP) allows for a hypertrophic response to insulin-like growth factor 1 (IGF-1) stimulation. With repetitive modules forming the bulk of nebulin’s structure, the SH3 and serine-rich domains in the C-terminus stand out. To study the functions of these domains we made a mouse model which eliminates both the serine-rich and the SH3 domains and mimics mutational effects found in patients. Pilot studies show that the model has widened Z-disk and muscle atrophy. Aim 2 will investigate the effects of nebulin’s serine-rich and SH3 domains on structure and function of skeletal muscle. To test NEM2 therapeutics in Aim 3 we made a mouse in which nebulin is conditionally deleted (Neb cKO) and that phenocopies NEM2. We will use this model to test the effects of gene therapy with adeno-associated delivery of functionally distinct subdomains of nebulin. We hypothesize that expressing the full Z-disk construct improves Z-disk integrity and hypertrophy signaling and that the effects are attenuated by eliminating the serine- rich and SH3 domains. We will also test whether expressing a SR construct enhances the force generating crossbridge population and will study the effects of Z-disk and SR constructs when expressed simultaneously. With its basic science and translational foci this proposal seeks to continue our track record of cutting edge nebulin research. We have a strong research team with excellent collaborators, use state of the art techniques, created novel mouse models for this work, and have supportive pilot data. We anticipate that this proposal will provide novel insights in the complex biology of nebulin and, importantly, how to ameliorate muscle weakness in nemaline myopathy.

Nebulin is a skeletal muscle protein that can cause the debilitating nemaline myopathy disease (NEM2) and this application is focused on investigating this poorly understood protein. We will investigate in novel mouse models the function of nebulin in thin filament length regulation, Z-disk structure, and hypertrophy signaling. Importantly, we will also study nebulin gene therapy as a means to restore muscle function in NEM2.