2013-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/642684摘要：橫紋肌是由慢速肌纖維和快速肌纖維所混合組成，生物可藉由調控慢速肌纖維與快速肌纖維的比例來達到反應及適應生理狀態的改變。由本實驗室所發現之核受器作用蛋白(Nuclear receptor interacting protein, NRIP)，目前研究已知在鈣離子存在下可與調鈣素(calmodulin)結合，並且進一步活化鈣調磷酸酶(calcineurin)之活性。近期報導指出，在臨床上發現肌強直性萎縮症患者缺乏NRIP基因的表現。我們的初期結果顯示，NRIP剔除鼠在滾輪、平面跑步機及橫隔膜收縮力測試實驗中均表現出肌肉強度不足之現象(Fig. 1 to 3)，代表著 NRIP 在調控肌肉強度中扮演著一定角色。另外，在酵母菌雙雜合系統(yeast two hybrid system)中發現α-actinin 2 是可與 NRIP 結合的蛋白質之一 (Fig. 4)，我們也在 in vitro 蛋白質結合實驗中進一步確認兩者有直接結合現象(Fig. 5)。因為α-actinin2是肌肉Z 線上的標定蛋白，並且α-actinin 2 與 Z 線上其他蛋白的結合調控著肌肉的功能，因此破壞這些蛋白的結合會造成肌肉功能失調。有趣的是我們發現在電顯結果中，NRIP剔除鼠的Z 線似乎比正常鼠之Z 線寬 (Fig. 7)，此結果暗示著 NRIP 可能參與Z 線的生成。因為骨骼肌與心肌皆為橫紋肌，因此 NRIP 可能與骨骼肌和心肌的發育階有關係。因此，我們推測 NRIP 可與α-actinin 2 在Z 線上結合，進一步調控影響橫紋肌肉功能相關之訊息路徑，如活化鈣調磷酸酶(calcineurin)訊息路徑和慢速肌凝蛋白(slow myosin)的表現等。在NRIP剔除鼠所發現之慢速肌凝蛋白(slow myosin) 核醣核酸(RNA)和蛋白質的表現皆下降(Fig. 8)可支持此推論。在本計畫中，我們將會持續利用已建立之NRIP剔除鼠進行有關 NRIP 在骨骼肌及心肌 Z 線上所參與之肌肉發育的功能。我們的遠程目標是發展 NRIP 成為治療肌肉萎縮症之標的。目前將會專注在骨骼肌上的療效，但NRIP也有可能在心臟扮演重要角色，因此未來本計畫也可能拓展到有關心肌功能之研究。<br> Abstract: The striated muscles are a mosaic of slow and fast twitch myofibers, and the relative number of slow- and fast-twitch fibers is tightly regulated to enable an organism to respond and adapt to altering physical conditions. We previously found a gene named nuclear receptor interaction protein (NRIP, Tsai et al., 2005, Chen et al., 2008) that is Ca+2-dependent calmodulin binding protein and then activate calcineurin phosphatase activity (now in revised). Recently it reported lack of NRIP gene expression in clinical muscular dystrophy (Zhang et al., 2006). Our preliminary results display that NRIP KO mice loss muscle strength from the assays of rotarod test (Fig. 1), treadmill test (Fig. 2) and diaphragm contraction test (Fig. 3); indicating NRIP role in muscle strength. Additionally, we find that alpha-actinin 2 is one of NRIP-interacting protein from yeast two hybrid systems (Fig. 4) and further confirmed the direct binding via in vitro protein-protein interaction (Fig. 5). Since alpha-actinin is biomarker of muscle Z-disc, interaction between Z-disc proteins regulates muscle functions and disruption of these interactions results in muscle dysfunction (Knöll et al., 2002). Intriguingly, we find that the band of Z-disc both in the skeletal and cardiac muscles of NRIP KO mice is wider than wt mice from electron photograph (magnification 30000 and 20000 respectively, Fig. 7A and 7B), implying that NRIP may involve in Z-disc formation. Due to skeletal and cardiac muscles belong to striated muscle; indicating that NRIP functions may involve in skeletal and cardiac striated muscle development. Therefore, we hypothesize that NRIP interacts with alpha-actinin 2 and locates in Z-disc (Fig. 6) which then activates the cascade calcineurin and slow myosin gene expression those regulating striated muscle function. It can be supported by the results of slow myosin RNA and protein expression are declined in NRIP KO mice compared to wild mice (Fig. 8). In this project, we will comprehensively investigate the Z-disc role of NRIP in skeletal and cardiac striated muscle development using NRIP knock out mice model. Our long-term goal is to determine NRIP as a therapeutic target for muscle dystrophy. As to therapeutic effect, here we will focus on skeletal muscle. However NRIP potentially plays an important role in heart. In the future, this project can extend to cardiomyopathy mechanism.心肌心臟衰竭慢速肌基因轉殖鼠