Specific knockdown of m-calpain blocks myogenesis with cDNA deduced from the corresponding RNAi.
Fusion of mononuclear myoblast to multinucleated myotubes is crucial for myogenesis. Both mu- and m-calpain are ubiquitously expressed in most cells and are particularly abundant in muscle cells. Knockout of calpain-1 (catalytic subunit of mu-calpain) induced moderate platelet dysaggregation, preserving the normal development and growth, although knockout of calpain-2 (m-calpain) is lethal in mice. Therefore, there should be muscle-specific function of m-calpain per se. Previous methods lack direct evidence for the involvement of m-calpain, because the specific inhibitor to m-calpain has not been developed yet and the inhibition was less potent. Here, we show that screened RNA interference (RNAi) specifically blocked the m-calpain expression by 95% at both the protein and the activity levels. After transfection of adenovirus vector-mediated cDNA corresponding to the RNAi-induced short hairpin RNA, m-calpain in C(2)C(12) myoblasts was knocked down with no compensatory overexpression of mu-calpain or calpain-3. The specific knockdown strongly inhibited the fusion to multinucleated myotubes. In addition, the knockdown modestly blocked ubiquitous effects, including cell migration, cell spreading, and alignment of central stress fiberlike structures. These results may indicate that m-calpain requiring millimolar Ca(2+) level for the full activation plays specific roles in myogenesis, independent of mu-calpain, and leave us challenging problems in the future.
Honda
Michiyo
M
Department of Chemistry, Faculty of Science and Engineering, Sophia University, Tokyo, Japan.
Masui
Fujiko
F
Kanzawa
Nobuyuki
N
Tsuchiya
Takahide
T
Toyo-oka
Teruhiko
T
D000818:Animals; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D002459:Cell Fusion; D002460:Cell Line; D002465:Cell Movement; D018076:DNA, Complementary; D005786:Gene Expression Regulation / Q000502:physiology; D051379:Mice; D024510:Muscle Development / Q000235:genetics / Q000502:physiology*; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D002154:Calpain; C449000:m-calpain; C569870:CAPN2 protein, human