The first goal of this study was to understand the role of calpains in skeletal muscle protein degradation in cultured cells. We have developed a genetic approach to inhibit endogenous calpain activity through over-expressing dominant negative m-calpain (DN), antisense m-calpain (AS) and calpastatin inhibitory domain (CID). We observed that, under conditions of accelerated degradation (serum withdrawal), inhibition of m-calpain through DN-m-calpain over-expression caused a 30% inhibition of total protein degradation whereas CID over-expression reduced degradation by 63%. These constructs did not significantly affect degradation in the presence of serum. These data indicate that calpains participate in the accelerated degradation associated with serum withdrawal. Inhibition of calpain also stabilized nebulin, a major structural protein of the sarcomere. These observations indicate that calpains play significant roles in muscle protein turnover. Finally, over-expression of antisense m-calpain caused a transient reduction in m-calpain concentration after which normal m-calpain concentration was quickly re-established. These observations indicate that m-calpain is a short half-life protein in muscle cells. The second goal of this study is to investigate the role of calpain in the mediation of PARP protein level in differentiating myoblasts. Poly(ADP-ribosyl)ation, catalyzed by PARP, is involved in various physiological events, such as DNA excision repair, DNA recombination, DNA replication, cell differentiation, cell growth and transformation, and apoptosis. A protease participating in PARP turnover could be a significant regulator to the events which PARP is involved. A relationship between apoptosis and myofibrillar protein degradation via a common protease might suggest the basis for muscle wasting and atrophy which characterize in many muscle diseases. We established a genetic approach to inhibit endogenous calpain activity through over-expressing calpastatin inhibitory domain (CID). We observed that (1) inhibition of calpain activity increased PARP concentration when post-confluent myoblasts were cultured with 2% HS medium, an inducer of differentiation and (2) inhibition of calpain activity prevented PARP degradation induced by A23187 and etoposide in differentiating myoblasts. These data demonstrate that calpain is involved in regulation of PARP in cultured cells. / Graduation date: 1998

Skeletal muscle proteins are the largest amino acid pool in animal body and are continuously degraded and resynthesized. Dozens of factors have been shown to influence the balance between synthesis and degradation and thereby regulate muscle growth and function. It is now know that one of the major regulatory bases of muscle metabolism is neuron-muscle interaction. A neurogenic factor, ciliary neurotrophic factor (CNTF), is proposed to exert myotrophic actions and could possible be a mediator of neuron-muscle interactions. The goal of this study was to investigate the effects of CNTF on muscle protein turnover in an in vitro system. CNTF was applied to differentiated cultured muscle cells (L8 cell line). Radiochemical labeled amino acids were added to the culture medium to determine the rate of incorporation or release by the cells as indexes of protein synthesis and protein degradation, respectively. Total protein was measured as an index of change in total protein accretion. Twelve hours of CNTF treatment increased myofibrillar protein synthesis by 10%. However, longer CNTF treatment (24 hours) reduced non-myofibrillar protein synthesis. CNTF (1 ng/ml) decreased protein degradation but higher doses (20 ng/ml) accelerated protein degradation. These changes in protein turnover resulted from changes in the myofibrillar protein fraction rather than from changes in turnover of the non-myofibrillar fraction. The change in protein synthesis and protein degradation resulted in an increase in total protein accretion of about 6%. Compared with the myotrophic studies on the effects of CNTF in vivo, the action of CNTF were relatively small. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that CNTF receptor alpha subunit (CNTFR��) mRNA expression is lower than which is expressed in muscle tissue. This could explain the reason why CNTF exerted smaller effects in vitro compared to those reported in vivo. Overall, CNTF exerts a small but statistically significant anabolic actions in cultured skeletal muscle and the actions were highly dose-dependent. The limited action of CNTF in vitro may be related to its low receptor density in the L8 cell (compared to in vivo). Because actions may be highly dose-dependent, a challenging series of studies are ahead for anyone wishing to identify the signal transduction mechanisms which account for CNTF's actions. / Graduation date: 1998

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. / Graduation date: 2002

Simon, Ming-yuen Lee. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 239-257). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

The loss of muscle mass leading to cachexia is rarely identified in early lung cancer. Fasting blood and muscle biopsy were collected in 59 non-small cell lung cancer (NSCLC) and 16 non-cancer patients, at the beginning of thoracic surgery. Serum C-reactive protein (CRP), and IL-6 were higher in NSCLC. In weight-losing NSCLC, food intake and serum albumin were lower, CRP, and TNF-alpha were higher. Although the expression of genes of the ubiquitin-proteasome system was not different, ubiquitinated-protein levels were lower and negatively correlated with ph-FOX01 in weight-losing patients. This would suggest lower muscle proteolytic rates in the early stages of NSCLC. Ph-FOXO1 also related to the degree of weight loss and stage of NSCLC. These data suggest that in early stages of the disease, weight and muscle loss could be mainly due to reduced food intake, rather than accelerated proteolysis, which reinforces the potential for successful dietary interventions to prevent or delay the onset of cachexia.

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