Beta-synemin expression in cardiotoxin-injected rat skeletal muscle.
BACKGROUND
Beta-synemin was originally identified in humans as an alpha-dystrobrevin-binding protein through a yeast two-hybrid screen using an amino acid sequence derived from exons 1 through 16 of alpha-dystrobrevin, a region common to both alpha-dystrobrevin-1 and -2. alpha-Dystrobrevin-1 and -2 are both expressed in muscle and co-localization experiments have determined which isoform preferentially functions with beta-synemin in vivo. The aim of our study is to show whether each alpha-dystrobrevin isoform has the same affinity for beta-synemin or whether one of the isoforms preferentially functions with beta-synemin in muscle.


METHODS
The two alpha-dystrobrevin isoforms (-1 and -2) and beta-synemin were localized in regenerating rat tibialis anterior muscle using immunoprecipitation, immunohistochemical and immunoblot analyses. Immunoprecipitation and co-localization studies for alpha-dystrobrevin and beta-synemin were performed in regenerating muscle following cardiotoxin injection. Protein expression was then compared to that of developing rat muscle using immunoblot analysis.


RESULTS
With an anti-alpha-dystrobrevin antibody, beta-synemin co-immunoprecipitated with alpha-dystrobrevin whereas with an anti-beta-synemin antibody, alpha-dystrobrevin-1 (rather than the -2 isoform) preferentially co-immunoprecipitated with beta-synemin. Immunohistochemical experiments show that beta-synemin and alpha-dystrobrevin co-localize in rat skeletal muscle. In regenerating muscle, beta-synemin is first expressed at the sarcolemma and in the cytoplasm at day 5 following cardiotoxin injection. Similarly, beta-synemin and alpha-dystrobrevin-1 are detected by immunoblot analysis as weak bands by day 7. In contrast, immunoblot analysis shows that alpha-dystrobrevin-2 is expressed as early as 1 day post-injection in regenerating muscle. These results are similar to that of developing muscle. For example, in embryonic rats, immunoblot analysis shows that beta-synemin and alpha-dystrobevin-1 are weakly expressed in developing lower limb muscle at 5 days post-birth, while alpha-dystrobrevin-2 is detectable before birth in 20-day post-fertilization embryos.


CONCLUSIONS
Our results clearly show that beta-synemin expression correlates with that of alpha-dystrobrevin-1, suggesting that beta-synemin preferentially functions with alpha-dystrobrevin-1 in vivo and that these proteins are likely to function coordinately to play a vital role in developing and regenerating muscle.
Mizuno
Yuji
Y
Department of Neurology, Gunma University Graduate School of Medicine, Showa, Maebashi, Gunma, Japan. mizunoy@med.gunma-u.ac.jp
Guyon
Jeffrey R
JR
Ishii
Akiko
A
Hoshino
Sachiko
S
Ohkoshi
Norio
N
Tamaoka
Akira
A
Okamoto
Koichi
K
Kunkel
Louis M
LM
D000818:Animals; D015153:Blotting, Western; D049029:Dystrophin-Associated Proteins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000096:biosynthesis*; D008297:Male; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000503:physiopathology; D009479:Neuropeptides / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
C490915:DTNA protein, human; C098884:Dtna protein, mouse; D049029:Dystrophin-Associated Proteins; D007381:Intermediate Filament Proteins; D009479:Neuropeptides; C430456:desmuslin