Deleting nebulin's C-terminus reveals its importance to sarcomeric structure and function and is sufficient to invoke nemaline myopathy

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 toward the thin filament pointed end. While nebulin's C-terminus has been implicated in both sarcomeric structure and function as well as the development of nemaline myopathy, the contributions of this region remain largely unknown. Additionally, the C-terminus is reported to contribute to muscle hypertrophy via the IGF-1 growth pathway. To study the functions of nebulin's C-terminus, we generated a mouse model deleting the final two unique C-terminal domains, the serine-rich region (SRR) and the SH3 domain (Neb [163][164][165] ). Homozygous Neb [163][164][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. Glutathione S-transferase (GST) pull-down experiments found novel binding partners with the SRR, several of which are associated with myopathies. While the C-terminus does not appear to be a limiting step in muscle growth, the IGF-1 growth pathway remained functional despite the deleted domains being proposed to be essential for IGF-1 mediated hypertrophy. The Neb 163-165 mouse model emphasizes that nebulin's C-terminus is necessary for proper sarcomeric development and shows that its loss is sufficient to induce myopathy.

Expressing a Z-disk nebulin fragment in nebulin-deficient mouse muscle: effects on muscle structure and function

“…1a). This AAV construct, estimated to be 139.9 kDa, was injected into the anterior compartment of the lower hindlimb of the mouse as previously described [31]. This allows for the AAV to enter muscle fibers and the construct to be expressed in both the tibialis cranialis (TC) and the extensor digitorum longus (EDL) muscles [59].…”

“…Fiber skinning was performed as previously described [31]. TC muscles were split and placed in relaxing solution (in mM: 40 BES, 10 EGTA, 6.56 MgCl 2 , 5.88 Na-ATP, 46.35 K-propionate, 15 creatine phosphate at pH 7.0) with 1% triton X-100 for demembranation (skinning).…”

“…Because of the Z-disk localization exhibited by the Zdisk fragment (above), and the previous finding that deleting a small portion of nebulin's Z-disk region alters Zdisk widths [31], the widths of the Z-disks in these samples were also analyzed (sarcomeres with nemaline rod bodies were avoided). While differences were difficult to discern even at high magnification (Additional file 1: Figure S3), an even sampling across multiple unique fibers of two mice within each treatment group revealed the changes in widths.…”

“…Outside of these modules, there is a glutamic acid-rich region at the N-terminus of the protein and two more unique domains at the C-terminus: the serine-rich region and the SH3 domain. While the glutamic acid-rich region remains unstudied, the serine-rich region and the SH3 domains are thought to contribute to the regulation of other sarcomeric proteins as well as the development of the Z-disk [31].…”

“…Due to nebulin's contributions to thin filament length regulation, force production, and structural maintenance [31][32][33][34][35][36][37][38][39][40][41], several studies have focused on improving these aspects of the sarcomere through therapeutic interventions. Studies targeting troponin activation have reported increases in force production at submaximal stimulation frequencies [42,43], but other attempts to improve muscle weight and function have shown that such therapeutic changes are difficult to achieve [44][45][46].…”