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First published online 4 December 2007
doi: 10.1242/jcs.020701

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Differential targeting of nNOS and AQP4 to dystrophin-deficient sarcolemma by membrane-directed -dystrobrevin

Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA

View larger version (46K): [in this window] [in a new window]   Fig. 1. Expression of transgenic -dystrobrevin in skeletal muscle. (A) The murine creatine kinase (MCK) promoter was used to drive expression of -dystrobrevin-2a linked to either HA at the C-terminus or to HA followed by the Ras palmitoylation site (RPS), to force membrane association. (B) Immunoblotting of muscle homogenates using an anti-dystrobrevin antibody show the relative transgene expression of the three founder lines of TgDB (63, 68, 80), five lines of TgDB-RPS (10, 17, 27, 29, 71) and wild type (wt). The bottom part of the same immunoblot was labeled with an antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to demonstrate that similar amounts of protein were loaded on the gel.

View larger version (41K): [in this window] [in a new window]   Fig. 2. Transgenic -dystrobrevin is functional when expressed in the -dystrobrevin-null mouse. (A) H&E-stained tibialis anterior (TA) muscle shows that transgene expression in the dystrobrevin-null mouse results in a more uniform fiber diameter and a reduction in the number of centrally nucleated fibers. (B) The number of centrally nucleated fibers in TA (dark shade), soleus (medium shade) and diaphragm (light shade) muscles are reduced by expression of each -dystrobrevin transgene. (C) Serum creatine kinase levels were likewise restored to normal with transgene expression. Data are from transgenic lines 63 (DB) and 29 (DB-RPS). Error bars give ± s.d. Scale bar, 50 µm.

View larger version (71K): [in this window] [in a new window]   Fig. 3. Restoration of -dystrobrevin-associated proteins by transgene expression. Sarcolemmal expression of -syntrophin and nNOS in TA muscle is reduced in the -dystrobrevin-null muscle but restored by expression of either the TgDB or TgDB-RPS. Data are from transgenic lines 63 (DB) and 29 (DB-RPS). Scale bar, 50 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 4. Transgenic -dystrobrevin expression in the mdx mouse. (A) H&E stained tibialis anterior (TA) muscle shows that the dystrophic phenotype persists in the mdx mice, despite the expression of either transgenic -dystrobrevin. (B) The number of centrally nucleated fibers in TA (dark shade), soleus (medium shade) and diaphragm (light shade) muscles are not significantly reduced by expression of either -dystrobrevin transgene. (C) Serum creatine kinase levels in mdx mice with or without transgenic -dystrobrevin are also similar. Error bars give ± s.d. Data was obtained from TgDB-RPS lines 29 and 71. Scale bar, 50 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 5. β-Dystroglycan and sarcoglycan expression in mdx mice expressing sarcolemmal -dystrobrevin. (A,B) Palmitoylated -dystrobrevin is specifically localized to the sarcolemma when expressed in the mdx mouse. However, the sarcolemmal -dystrobrevin fails to restore normal levels of either β-dystroglycan or -sarcoglycan, as determined by (A) immunofluorescence of TA muscle or (B) immunoblots of membrane proteins. Data were obtained from TgDB-RPS line 29. Scale bar, 50 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 6. Select restoration of dystrophin associated proteins by sarcolemmal -dystrobrevin. (A,B) Palmitoylated -dystrobrevin expressed on the sarcolemma of TA muscle of mdx mice restored the levels of -syntrophin and AQP4 but failed to restore sarcolemmal nNOS as determined by (A) immunofluorescence and (B) immunoblots of membrane proteins. Sarcolemmal utrophin expression was not altered by sarcolemmal -dystrobrevin. Data was obtained from TgDB-RPS lines 29 and 71. Scale bar, 50 µm.

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