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.