Nesprin 1 is an outer nuclear membrane protein that is thought to link the nucleus to the actin cytoskeleton. Recent data suggest that mutations in Nesprin 1 may also be involved in the pathogenesis of Emery-Dreifuss muscular dystrophy. To investigate the function of Nesprin 1 in vivo, we generated a mouse model in which all isoforms of Nesprin 1 containing the C-terminal spectrin-repeat region with or without KASH domain were ablated. Nesprin 1 knockout mice are marked by decreased survival rates, growth retardation and increased variability in body weight. Additionally, nuclear positioning and anchorage are dysfunctional in skeletal muscle from knockout mice. Physiological testing demonstrated no significant reduction in stress production in Nesprin 1-deficient skeletal muscle in either neonatal or adult mice, but a significantly lower exercise capacity in knockout mice. Nuclear deformation testing revealed ineffective strain transmission to nuclei in muscle fibers lacking Nesprin 1. Overall, our data show that Nesprin 1 is essential for normal positioning and anchorage of nuclei in skeletal muscle.

SUMMARY The use of sarcomere length to normalize fiber length in architectural studies is commonly practiced but has not been explicitly validated. Using mouse hindlimb muscles as a model system, ankle joints were intentionally set to angles ranging from 30° to 150° and their muscles fixed. Tibialis anterior (TA), extensor digitorum longus (EDL) and soleus muscles were removed and their raw fiber length measured. Sarcomere length was then measured for each fiber length sample and fiber length was normalized to a standard sarcomere length. As expected, raw fiber length was dependent on tibiotarsal angle (P<0.0005 for all muscles, r2 range 0.22–0.61), while sarcomere length normalization eliminated the joint-angle dependent variation in fiber length (P>0.24, r2 range 0.001–0.028). Similarly, one-way ANOVA revealed no significant differences in normalized fiber length among ankle angles for any of the three muscles (P>0.1), regardless of animal size. To determine the resolution of the method, power calculations were performed. For all muscles studied, there was >90% chance of detecting a 15% fiber length difference among muscles and >60% chance of detecting fiber length differences as small as 10%. We thus conclude that the use of sarcomere length normalization in architectural studies permits resolution of fiber length variations of 15% and may even be effective at resolving 10%fiber length variations.

The development and function of skeletal muscle depend on molecules that connect the muscle fiber cytoskeleton to the extracellular matrix (ECM). β1 integrins are ECM receptors in skeletal muscle, and mutations that affect the α7β1 integrin cause myopathy in humans. In mice, β1 integrins control myoblast fusion, the assembly of the muscle fiber cytoskeleton, and the maintenance of myotendinous junctions (MTJs). The effector molecules that mediate β1 integrin functions in muscle are not known. Previous studies have shown that talin 1 controls the force-dependent assembly of integrin adhesion complexes and regulates the affinity of integrins for ligands. Here we show that talin 1 is essential in skeletal muscle for the maintenance of integrin attachment sites at MTJs. Mice with a skeletal muscle-specific ablation of the talin 1 gene suffer from a progressive myopathy. Surprisingly, myoblast fusion and the assembly of integrin-containing adhesion complexes at costameres and MTJs advance normally in the mutants. However, with progressive ageing, the muscle fiber cytoskeleton detaches from MTJs. Mechanical measurements on isolated muscles show defects in the ability of talin 1-deficient muscle to generate force. Collectively, our findings show that talin 1 is essential for providing mechanical stability to integrin-dependent adhesion complexes at MTJs, which is crucial for optimal force generation by skeletal muscle.