Nebulin is a giant filamentous protein found in skeletal muscle where it spans from the sarcomere's Z-disk to near the tip of the thin filament. The long-term goal of this proposal is to gain a mechanistic understanding of the roles of nebulin in skeletal muscle health and disease. Nebulin's functions have remained largely obscure until the recent development of a nebulin knockout (NEB KO) mouse model that has greatly accelerated progress. Recent results established that nebulin plays structural roles in thin filament length control, but, unexpectedly, also suggest that nebulin might be important in muscle contraction by increasing the level of thin filament activation at submaximal calcium levels and by altering crossbridge cycling kinetics to increase the fraction of crossbridges that develops force. This is an exciting new concept not only because it reveals a previously unknown major aspect of skeletal muscle physiology, but also because it might help to explain why nemaline myopathy (NM) patients with nebulin deficiency have severe muscle weakness. The role of nebulin in muscle contraction will be intensely examined and thoroughly tested in this proposal.
Aim 1 will study the sarcomere length (SL) and fiber type dependence of nebulin's effect on calcium sensitivity and crossbridge cycling kinetics. This work includes study of force kinetics in single myofibrils and the effect of nebulin on crossbridge recruitment. Similar to tropomyosin(Tm), nebulin occupies different sites on F-actin and the hypothesis that nebulin acts in concert with Tm to activate the thin filament will be studied in Aim 2 by using X-ray diffraction studies on wildtype (WT) and NEB KO muscle. This work will also focus on additional structural parameters including thin filament compliance, which based on single molecule studies has been suggested to be affected by nebulin and to function as a mechanism for controlling crossbridge kinetics. A prominent group of nemaline myopathy (NM) patients has nebulin deficiency that results from an in- frame deletion of nebulin's exon 55 and for the present proposal a novel mouse disease model was made in which exon 55 (NEB ?ex55) has been deleted. Recently the first homozygous mice were obtained and their preliminary analysis revealed multiple similarities between the mice and NM patients, including severe muscle weakness.
Aim 3 will determine the functional properties of this model at the intact muscle, skinned fiber, and myofibrillar levels and compare results to those on biopsies from NM patients. To gain insights into therapies that might augment contractile strength in nebulin-deficient skeletal muscle (mouse models and NM patients) the effect of calcium sensitizers will be studied in Aim 4. The innovation of this proposal lies in the novel research foci with innovative guiding hypotheses, its novel mouse models of human disease, and its novel experimental tools. The proposal's integrative approach is expected to lead to an in-depth understanding of the roles of nebulin in skeletal muscle function and to greatly increase understanding of the mechanisms that give rise to muscle weakness in nemaline myopathy, and to provide therapeutic insights.
Severe muscle weakness is a characteristic of patients with the neuromuscular disorder nemaline myopathy (NM). Most NM patients have mutations in the giant protein nebulin that result in low amounts or non-functional nebulin. We focus on the recent discovery that nebulin is needed for normal force levels during muscle contraction. The proposed work consists of cutting-edge muscle biophysics and X-ray structural analyses and includes studies on a mouse model that we made that mimics an important nebulin mutation found in NM patients. By comparing the model to patients we will establish whether the mouse model is a platform for studying disease mechanisms and testing therapeutics. As an example of a therapeutic for increasing muscle strength we study calcium sensitizers.
|Gineste, C; De Winter, J M; Kohl, C et al. (2013) In vivo and in vitro investigations of heterozygous nebulin knock-out mice disclose a mild skeletal muscle phenotype. Neuromuscul Disord 23:357-69|
|Lee, Eun-Jeong; De Winter, Josine M; Buck, Danielle et al. (2013) Fast skeletal muscle troponin activation increases force of mouse fast skeletal muscle and ameliorates weakness due to nebulin-deficiency. PLoS One 8:e55861|
|Ottenheijm, Coen A C; Buck, Danielle; de Winter, Josine M et al. (2013) Deleting exon 55 from the nebulin gene induces severe muscle weakness in a mouse model for nemaline myopathy. Brain 136:1718-31|
|de Winter, Josine Marieke; Buck, Danielle; Hidalgo, Carlos et al. (2013) Troponin activator augments muscle force in nemaline myopathy patients with nebulin mutations. J Med Genet 50:383-92|
|Labeit, Siegfried; Ottenheijm, Coen A C; Granzier, Henk (2011) Nebulin, a major player in muscle health and disease. FASEB J 25:822-9|
|Ottenheijm, Coen A C; Lawlor, Michael W; Stienen, Ger J M et al. (2011) Changes in cross-bridge cycling underlie muscle weakness in patients with tropomyosin 3-based myopathy. Hum Mol Genet 20:2015-25|
|Ottenheijm, Coen A C; Hooijman, Pleuni; DeChene, Elizabeth T et al. (2010) Altered myofilament function depresses force generation in patients with nebulin-based nemaline myopathy (NEM2). J Struct Biol 170:334-43|
|Ottenheijm, Coen A C; Granzier, Henk (2010) New insights into the structural roles of nebulin in skeletal muscle. J Biomed Biotechnol 2010:968139|
|Buck, Danielle; Hudson, Bryan D; Ottenheijm, Coen A C et al. (2010) Differential splicing of the large sarcomeric protein nebulin during skeletal muscle development. J Struct Biol 170:325-33|
|Tonino, Paola; Pappas, Christopher T; Hudson, Bryan D et al. (2010) Reduced myofibrillar connectivity and increased Z-disk width in nebulin-deficient skeletal muscle. J Cell Sci 123:384-91|
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