Nebulin, a giant and abundant muscle protein, is the fourth myofilament of the skeletal muscle sarcomere. Its name is derived from the early realization that its functions are unclear (nebulous), which remains true to date. To elucidate the functions of nebulin in vivo we created a nebulin KO mouse model. KO mice survive for up to several weeks and preliminary data show that their skeletal muscles are amenable to physiological and structural studies. We propose to test the roles of nebulin in thin filament activation and crossbridge kinetics, Z-disk structure, and thin filament length control. We also aim to resolve whether nebulin plays a significant role in thin-filament length regulation of cardiac muscle and will study the role of nebulin in patients with nemaline myopathy. Our preliminary skinned muscle fiber studies indicate that regulation of contraction is significantly altered in nebulin KO mice and that maximal active tension is greatly reduced. Our preliminary studies of KO animals also reveal pathological Z-disk structures, similar to those described in nemaline myopathy patients. Using several ultra-structural approaches we obtained preliminary evidence that thin-filament length control is lost in skeletal muscle of KO animals. Further, KO mice upregulate sarcolipin, indicating that the SR calcium uptake capacity is reduced in intact muscle. Work on NM patients provides evidence for perturbed capping functions of mutant nebulin, and reduced contractility. The proposed work will critically examine nebulin's role in the structure and function of the Z-disk, thin filament length control, and muscle contraction. We will extensively use the nebulin KO model and in addition we will carry out 'rescue experiments'. We anticipate that the proposed research will significantly increase understanding of the role of nebulin in striated muscle. Novel insights in disease mechanisms at play in nemaline myopathy are also expected. Finally, the work will establish whether nebulin plays a significant role in thin-filament length regulation of cardiac muscle. Essential to the proposed work is that we have available a mouse model in which the nebulin gene has been inactivated and that extensive preliminary data on both the nebulin KO mouse and patients with nemaline myopathy support the specific aims. ? ? ? ?
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