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Nemaline myopathy (NM) is a congenital myopathy with an estimated incidence of 1:50,000 live births. It is caused by mutations in thin filament components, including nebulin, which accounts for about 50% of the cases. The identification of NM cases with nonsense mutations resulting in loss of the extreme C-terminal SH3 domain of nebulin suggests an important role of the nebulin SH3 domain, which is further supported by the recent demonstration of its role in IGF-1-induced sarcomeric actin filament formation through targeting of N-WASP to the Z-line. To provide further insights into the functional significance of the nebulin SH3 domain in the Z-disk and to understand the mechanisms by which truncations of nebulin lead to NM, we took two approaches: (1) an affinity-based proteomic screening to identify novel interaction partners of the nebulin SH3 domain; and (2) generation and characterization of a novel knockin mouse model with a premature stop codon in the nebulin gene, eliminating its C-terminal SH3 domain (NebDSH3 mouse). Surprisingly, detailed analyses of NebDSH3 mice revealed no structural or histological skeletal muscle abnormalities and no changes in gene expression or localization of interaction partners of the nebulin SH3 domain, including myopalladin, palladin, zyxin and N-WASP. Also, no significant effect on peak isometric stress production, passive tensile stress or Young's modulus was found. However, NebDSH3 muscle displayed a slightly altered force-frequency relationship and was significantly more susceptible to eccentric contraction-induced injury, suggesting that the nebulin SH3 domain protects against eccentric contraction-induced injury and possibly plays a role in finetuning the excitation-contraction coupling mechanism. [ABSTRACT FROM AUTHOR]

Nebulin (NEB) is a large, rod-like protein believed to dictate actin thin filament length in skeletal muscle. NEB gene defects are associated with congenital nemaline myopathy. The functional role of NEB was investigated in gastrocnemius muscles from neonatal wild-type (WT) and NEB knockout (NEB-KO) mice, whose thin filaments have uniformly shorter lengths compared with WT mice. Isometric stress production in NEB-KO skeletal muscle was reduced by 27% compared with WT skeletal muscle on postnatal day 1 and by 92% on postnatal day 7, consistent with functionally severe myopathy. NEB-KO muscle was also more susceptible to a decline in stress production during a bout of 10 cyclic isometric tetani. Length-tension properties in NEB-KO muscle were altered in a manner consistent with reduced thin filament length, with length-tension curves from NEB-KO muscle demonstrating a 7.4% narrower functional range and an optimal length reduced by 0.13 muscle lengths. Expression patterns of myosin heavy chain isoforms and total myosin content did not account for the functional differences between WT and NEB-KO muscle. These data indicate that NEB is essential for active stress production, maintenance of functional integrity during cyclic activation, and length-tension properties consistent with a role in specifying normal thin filament length. Continued analysis of NEB's functional properties will strengthen the understanding of force transmission and thin filament length regulation in skeletal muscle and may provide insights into the molecular processes that give rise to nemaline myopathy. [ABSTRACT FROM AUTHOR]