Regulation of skeletal muscle protein degradation by u-calpain and development of a skeletal muscle-specific inducible expression system Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/qb98mj83m

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  • The first goal of this study was to understand the role of u-calpain in skeletal muscle protein degradation in cultured muscle cells. Several strategies were developed to down-regulate endogenous u-calpain activity and m-calpain activity in rat myotubes. These included over-expression of antisense u-calpain (AnsL), dominant negative u-calpain (DN-u-CL), antisense 30K subunit (AnsS) and fused antisense u-calpain/30K (AnsLS, i.e., 80K/30K). The ability to regulate calpain activity was confirmed by fodrin degradation (an index of calpain activity). Our data supported the contention that u-calpain contributes significantly to total protein degradation in myotubes. Specifically, over-expressing DN-u-calpain reduced total protein degradation by 7.9% (P<0.01) at 24 hr time point and by 10.6% (P<0.01) at a 48 hr time point. Similarly, over-expression of antisense u-CL and the 30K subunit reduced total protein degradation significantly at the 24 hr time point (P<0.05). However, over-expression of the fused antisense (80K/30K) did not affect (P>0.05) the total protein degradation. In addition to this we determined that desmin was a calpain substrate and that calpain could not degrade tropomyosin. The second goal of this study was to evaluate the relationships among u- and m-calpain and the 30KD subunit. The rationale for this study was that our earlier work indicated coordinated regulation of the calpain subunits. Our data demonstrated for the first time that the transcription and translation of u-calpain and 30K, and m-calpain and 30K are coordinately regulated, respectively. However, the expression of u-calpain did not affect the expression of m-calpain The third goal of this study was to develop a skeletal muscle-specific inducible expression system that may be used in transgenic animal research. A skeletal muscle a-actin promoter was used to replace the cytomegalovirus immediate-early promoter (pCMV) in the ecdysone inducible mammalian expression system. LacZ was used as a reporter gene. A beta-galactosidase staining assay and high-sensitivity B-gal activity assay indicated that the skeletal muscle-specific expression system functioned in myotubes. After 48 hr of administration of ponasterone A (inducer), the treated cells had 15-fold higher B-gal activity than the control cells.
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