| Budget Amount *help |
¥5,800,000 (Direct Cost: ¥5,800,000)
Fiscal Year 2004: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2003: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2002: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2001: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
It is known that dystrophin is an important protein in muscle biology to form the complex with other proteins (dystrophin complex). However, recent gene technique revealed that many other proteins are capable of causing muscular dystrophies, for example caveolin-3 gene mutation produces limb-girdle muscular dystrophy type 1C. Therefore, I have examined the proteins interacted with dystrophin complex in skeletal and cardiac muscle, and focused on following points : (1) caveolin-3, (2) nitric oxide, and (3) neuregulin. (1) Previous studies reported that caveolin-3 is associated with dystrophin complex. Caveolin-3 is thought to serve as a structural and a scaffolding protein in caveolae of the striated muscle. However, its precise localization in cardiac muscle has not been determined. Confocal laser microscopy and immunoelectron microscopy demonstrated that caveolin-3 was localized at T-tubule. Moreover, caveolin-3 was coprecipitated with alpha-actinin in cardiac muscle extraction but no
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t in skeletal muscle. These data raise the hypothesis that caveolin-3 serves as a mediator between sarcomeric dynamics via Z-bands and T-tubule function in cardiac muscle. (2) To reveal the caveolin-3 function, we examined endothelial nitric oxide synthase (eNOS). Since nitric oxide (NO), produced by nitric oxide synthases (NOSs), serves as many physiological properties in many cells. Immunoelectron micrographs clearly showed that eNOS was contained in sarcoplasmic reticulum (SR) encompassing Z-bands in addition to terminal cisternae in skeletal muscle, and expressed in caveolae, T-tubule, and SR in cardiac muscle. Immunoblotting using sucrose-gradient centrifugation showed that eNOS is detected in the SR fraction of both muscle. Furthermore, eNOS and caveolin-3 is coprecipitated in cardiac muscle extraction. These findings indicate that eNOS is constitutively expressed in SR of mouse skeletal and cardiac muscle fiber, and raises the hypothesis that NO derived from eNOS may play a role in muscle contraction. (3) Previous studies have indicated that ErbB receptors for neuregulins play a very important role in cardiac development, and neuregulin-ErbB signaling stimulates skeletal muscle regeneration. In addition, recent therapy for breast cancer with humanized monoclonal ErbB2 antibody has faced the problem of inducing cardiomyopathy. So, it is very intriguing and pivotal to investigate this signaling effect on both muscles. Immunoelectron micrographs clearly demonstrated ErbB2 locating at T-tubule in both skeletal and cardiac muscle, suggesting that neuregulin-ErbB2 signaling plays a role in the physiological function of cardiac and skeletal muscle even in adulthood.
Near the future, based on these findings, I will examine the skeletal and cardiac muscle of mdx mice which have been widely used as a model for Duchenne muscular dystrophy because of lacking dystrophin to unclose the pathological mechanism for muscular dystrophy and to develop a new therapy for it. Less
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