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This Article Full Text Full Text (PDF) Supplemental Figure All Versions of this Article: 294/4/C957    most recent 00505.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Honda, M. Articles by Toyo-oka, T. Search for Related Content PubMed PubMed Citation Articles by Honda, M. Articles by Toyo-oka, T.

MUSCLE CELL BIOLOGY AND CELL MOTILITY

Specific knockdown of m-calpain blocks myogenesis with cDNA deduced from the corresponding RNAi

¹Department of Chemistry, Faculty of Science and Engineering, Sophia University, Tokyo; ²Department of Organ Pathophysiology and Internal Medicine, University of Tokyo, Tokyo; and ³Department of Molecular Cardiology, Division of Biofunctional Sciences, Tohoku University Bioengineering Organization (TUBERO), Sendai, Japan

Submitted 25 October 2007 ; accepted in final form 19 January 2008

Fusion of mononuclear myoblast to multinucleated myotubes is crucial for myogenesis. Both µ- and m-calpain are ubiquitously expressed in most cells and are particularly abundant in muscle cells. Knockout of calpain-1 (catalytic subunit of µ-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 C₂C₁₂ myoblasts was knocked down with no compensatory overexpression of µ-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 Ca²⁺ level for the full activation plays specific roles in myogenesis, independent of µ-calpain, and leave us challenging problems in the future.

RNA interference; muscle cell development; fusion; adenovirus vector