G.P.147

Nesprin proteins have a central rod domain of spectrin repeats and are intracellular linkers and scaffolds. Full length or “giant” nesprin-1 and nesprin-2 have N-terminal calponin homology domains that bind the actin cytoskeleton and C-terminal transmembrane KASH domains which anchor the nesprins to the outer nuclear membrane. A number of short isoforms of nesprin-1 and nesprin-2 are produced by internal promotion and by alternative splicing. Mutations in nesprin-1 and nesprin-2 can cause Emery-Dreifuss muscular dystrophy and dilated cardiomyopathy. Nesprin-1-alpha-2 is found almost exclusively in skeletal muscle and heart and all the known mutations in nesprins that are associated with EDMD or DCM lie within the alpha isoform sequence. Two novel “epsilon” isoforms of nesprin-2 were predicted by bioinformatics and we have demonstrated the existence of corresponding proteins for the first time. Because they are similar to nesprin-1 alpha in size and structure, we evaluated the significance of the epsilon isoforms in 20 human tissues and 7 human cell lines using qPCR. N2-epsilon-1 was expressed only in ovary and early embryonic cells (Ntera-2 and ESC), while N2-epsilon-2 was expressed in several mature tissues, including cardiac, but not skeletal, muscle. Western blotting confirmed the presence of epsilon-1 protein in Ntera-2 and ESC and epsilon-2 protein in heart and brain. Total nesprin transcript in ESC was mainly nesprin-2-giant (77%) and nesprin-2-epsilon-1 (21%). PCR indicated that most of the nesprin-2 mRNA in ESC lacked the KASH domain, whereas heart and skeletal muscle appeared to contain high levels of nesprin-2 KASH. Immunofluorescence microscopy with a monoclonal antibody against nesprin-2 showed nuclear rim staining in heart and skeletal muscle sections, but in KASH-less ESC there was an intense nucleoplasmic speckle-like distribution. Supported by British Heart Foundation (PG/11/71/29091).