Specific knockdown of delta-sarcoglycan gene in C2C12 in vitro causes post-translational loss of other sarcoglycans without mechanical stress.
The precise role of delta-sarcoglycan (SG) that is constitutively expressed in skeletal muscle cells and may serve for maintaining the sarcolemmal integrity has not been identified. The delta-SG protein is at first among SG complex. To specifically identify the role in C(2)C(12) cells during the myogenesis, we screened several RNA interference (RNAi) candidates at first, and knocked down both levels of the mRNA and protein, employing adenovirus-mediated RNAi. We found no morphological alteration at both myoblast and myotube stages by suppression of delta-SG. The specific knockdown of delta-SG accompanied a concomitant decrease of alpha-, beta-, and gamma-SGs preserving normal levels of each transcript. As for the localization, alpha-, beta-, and gamma-SGs were weakly stained on the cell membrane in delta-SG knockdown cells, whereas each SG in control cell was localized both on the cell membrane and myoplasm abundantly. This enhanced post-translational loss would represent similitude of the progression of cardiomuscular diseases in vitro. Different from cardiac muscle cells, skeletal muscle cell culture without muscle contraction may imply that mechanical stress per se is not primarily involved in the progression of limb-girdle muscular dystrophy. Furthermore, we have observed translocation of calpain-2 to cell membrane in delta-SG knockdown cells, suggesting that Ca(2+)-sensitive proteases, calpains closely take part in post-translational proteolysis.
Honda
Michiyo
M
Department of Materials and Life Science, Faculty of Science and Engineering, Sophia University, Tokyo, 102-8554, Japan. honda-m@sophia.ac.jp
Hosoda
Mari
M
Kanzawa
Nobuyuki
N
Tsuchiya
Takahide
T
Toyo-oka
Teruhiko
T
D000818:Animals; D002154:Calpain / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D024510:Muscle Development / Q000502:physiology; D049288:Muscular Dystrophies, Limb-Girdle / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D049031:Sarcoglycans / Q000235:genetics* / Q000378:metabolism; D013314:Stress, Mechanical
D020033:Protein Isoforms; D049031:Sarcoglycans; D002154:Calpain