The role of nucleo-cytoskeletal link proteins in skeletal muscle

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DESCRIPTION (provided by applicant): Recent data suggest that mutations in Nesprin 1 and 2 may be involved in the pathogenesis of Emery-Dreifuss muscular dystrophy (EDMD), which is characterized by progressive skeletal muscle weakness with associated muscle contractures, and variable cardiac defects. 40% of patients with EDMD have been shown to have mutations in Emerin or Lamin A/C, two genes encoding proteins localized to the inner nuclear membrane (INM) and its underlying lamina, respectively. The INM, the outer nuclear membrane (ONM), and the nuclear lamina comprise the nuclear envelope, which is linked to the cytokeleton by members of both the SUN and Nesprin protein families. Approximately 60% of EDMD patients do not have mutations in either Emerin or LMNA. Intriguingly, mutations in Nesprin 1 and 2 have been associated with EDMD. Nesprins belong to a newly discovered family of mammalian spectrin-repeat proteins. Mice lacking Lamin A/C exhibit features of EDMD. Studies on skeletal muscle cultures isolated from these mice have demonstrated a critical role for Lamin A/C in skeletal myoblast differentiation and in mechanical stiffness by maintaining nucleo-cytoskeletal integrity. In contrast, Emerin knockout mice do not exhibit EDMD. However, in skeletal muscle cultures from Emerin null mice, myoblast differentiation is perturbed to the same extent as observed in Lamin A/C null cells. Moreover, although Emerin null cells do not exhibit decreased mechanical stiffness as found in Lamin A/C null cells, perturbations in gene expression associated with mechanotransduction are observed. Both Nesprin 1 and 2 are ubiquitously expressed. To investigate the functional roles of Nesprin 1 and 2, we have generated floxed alleles for Nesprin 1 and 2. By utilizing protamine Cre mice, we have generated global loss of function mutants for Nesprin 1 and 2 (Nesprin 1-/- and Nesprin 2-/-). Our studies reveal that Nesprin 2-/- mice are viable and have no obvious basal phenotype, whereas approximately 60% of Nesprin 1-/- mice die perinatally. Remaining survivors have reduced body weight and compromised exercise capacity. We also found 100% perinatal lethality in Nesprin 1-/-;Nesprin 2-/- double mutant mice. Histological analyses of Nesprin 1-/- mice and Nesprin 1-/-;Nesprin 2-/- mice revealed abnormal positioning of non-synaptic nuclei and disappearance of clusters of synaptic nuclei. The overall goals of this proposal are to test the hypothesis that Nesprin 1 and 2 have distinct and overlapping roles in skeletal muscle nuclear positioning, nuclear membrane integrity, skeletal myoblast differentiation, mechanical stiffness, mechano-transduction, and muscle function. We will achieve these goals by comprehensive molecular, biochemical, histological, and physiological analysis of the skeletal muscle phenotypes in our four existing genetically engineered mouse lines. Results will shed light into mechanisms by which mutations in Nesprin contribute to myopathies.