摘要:我們之前找到的基因-核接受子結合蛋白(Nuclear receptor interaction protein, NRIP)為一在鈣離子存在下可與攜鈣素(Calmodulin, CaM)結合並活化鈣調磷酸酶(Calcineurin, CaN)活性的蛋白。鈣調磷酸酶是肌纖維中Z 複合體的一員。我們初步研究成果顯示利用酵母菌雙雜合系統發現α-輔肌動蛋白2 型(α-actinin 2, ACTN2)可與NRIP 結合並利用體外或體內蛋白結合測試進一步確定,也利用免疫螢光染色測定其共同座落於Z 複合體上 (Fig2)。由於α-輔肌動蛋白2 型是組成肌肉Z 複合體結構的重要蛋白,Z 複合體蛋白間的相互作用調控著心臟的功能而這些相互作用的瓦解也時常導致心臟衰竭的發生。我們已成功培育出全身性的NRIP 基因剔除鼠(NRIP KO)(Fig. 1),有趣的是從電子顯微觀察發現NRIP 基因剔除鼠的骨骼肌與心肌中的Z 複合體均比正常老鼠來得寬 (Fig.3),我們初步的成果也顯示在滾輪測試及肌力體外測試中,NRIP 基因剔除鼠均有肌力缺乏的現象(Fig. 4),顯示NRIP 在肌力表現上扮演角色。此外,利用極度踏車運動試驗測試也顯示NRIP 基因剔除鼠的心肌耐力要比正常老鼠來得弱;非侵入式心臟超音波檢測也可測出NRIP 基因剔除鼠的心肌短縮分率(Fractional shortening, FS)以及心室收縮舒張內徑比(ESD/EDD)較低 (Fig. 5),及年老的NRIP 基因剔除小鼠有心臟肥大及心臟組織纖維化的情形 (Fig. 6). 由於骨骼肌與心肌同屬橫紋肌,顯示NIRP 的功能可能參與在橫紋肌發育上。因此在此計畫中,我們將利用NRIP 的Flox 老鼠與揭開肌酸(Creatine)磷酸酶所控制的Cre 老鼠交配培育NRIP 的肌肉特定基因剔除鼠,加上NRIP 的基因轉殖鼠專注並且深入探討解剖學及組織學上NRIP 在骨骼肌及心肌功能的角色。同時,我們利用遺傳譜系研究追蹤不同組織中特別是骨骼肌、心肌、大腦及小腦中高度表現NRIP 細胞的功能(Fig. 8),因此我們將培育R26R/NRIP-CreERT2 基因轉殖鼠,此鼠將可在抗雌激素的誘導下表現Cre 於表現NRIP 的組織中調控表現NRIP 的細胞。除此之外,利用特別感染於骨骼肌與心肌的AAV8 型夾帶NRIP 基因來測試其對NRIP 基因剔除鼠肌肉收縮功能、心臟超音波檢測的補救效果。我們的長期目標為測定NRIP 為肌肉萎縮及心肌症的治療標的。具體目標:1. 培育肌肉組織特定揭開肌酸磷酸酶之調控的Cre 老鼠,以得到特定於肌肉的基因剔除鼠,分析骨骼肌與心肌發育及功能。2. 培育NRIP 的基因轉殖鼠並研究其於骨骼肌與心肌發育的影響。3. 以微陣列分析比較基因剔除鼠與正常鼠在肌肉部分基因表現的差異。4. 培育含NRIP 啟動子的R26R/NRIP-CreERT2 品系老鼠以利用遺傳譜系追蹤有NRIP表現的細胞功能。5. 利用對骨骼肌或心肌有感染偏好性的AAV 第8 型攜帶NRIP 基因感染NRIP 的基因剔除鼠,使用肌力收縮測試及心臟超音波觀察其補救效果。
Abstract: We previously found a gene named nuclear receptor interaction protein (NRIP, Tsai et al.,2005, Chen et al., 2008) that is calcium-dependent calmodulin binding protein (Ca+2/CaM)and then activate calcineurin phosphatase activity (Chang et al., 2011). Calcineurin is a familyof sarcomeric Z-Disc. Now we successfully generated NRIP knock out mice (NRIP KO; Fig.1). Our preliminary results find that alpha-actinin 2 is one of NRIP-interacting protein fromyeast two hybrid systems and further confirmed the direct binding via in vitro protein-proteininteraction and colocalize in Z disc by immunofluorescence assay (Fig 2). Since alpha-actininis biomarker of muscle Z-disc, interaction between Z-disc proteins regulates heart functionsand disruption of these interactions results in heart failure. Intriguingly, we find that theZ-disc of NRIP KO mice in the skeletal muscle is wider than wt mice; plus the Z-disc incardiac muscles of NRIP KO is disorder from electron photograph (magnification 30000 and20000 respectively, Fig. 3A and 3B). Additionally we also showed that NRIP KO mice lossmuscle strength from the assays of rotarod test (Fig. 4A) and in vitro muscle contraction testwith neuromuscular blocker to rule out the nerve effect on muscle contraction (Fig. 4B, 4C);indicating NRIP role in muscle strength. Additionally, cardiac stress test by treadmill assays inKO mice shows weaker than wt mice (Fig. 5A); and heart functions by non-invasiceultrasonic heart image assays (Echocardiography) in NRIP KO mice show the diminishedfractional shortening [FS] and the ration of end-systolic diameter to end-diastolic diameter[ESD/EDD]) (Fig. 5B). Furthermore, NRIP-deficient mice exhibit cardiac hypertrophy andfibrosis with ageing (Fig. 6). Due to both skeletal and cardiac muscles belong to striatedmuscle; indicating that NRIP functions may involve in skeletal and cardiac striated muscledevelopment. Hence in this project, we will specifically and extensively investigate thedetailed anatomy and histology studies of NRIP in skeletal and cardiac striated musclefunctions by generating muscle specific NRIP KO mice using NRIP floxed mice (Fig. 1A)crossing with creatine kinase-driven Cre mice; plus NRIP knock-in transgenic mice.Simultaneously we will use genetic lineage tracing studies to pursuit the function ofNRIP-expressing cells in different tissues especially skeletal, cardiac, cerebrum andcerebellum where are highly express NRIP (Fig. 8), hence the R26R/NRIP-CreERT2transgenic mice will be generated and NRIP-expressing cells can be regulated by tamoxifen(TAM)-inducible expression of Cre in NRIP-expression tissues (Tian et al., 2011).Additionally, rescue effect of NRIP KO mice by AAV-8 promoter driven NRIP (specific tropicskeletal and heart muscles) will be examined by muscle contraction assays, Echocardiographand pathogenesis analysis. Our long-term goal is to determine NRIP as a therapeutic target formuscular dystrophy and cardiomyopathy. The following specific aims will be approached.Based on the above results, we applied the patent entitled “METHOD FOR MODULATINGSLOW MYOSIN” both in Taiwan and United States and now is in process.Specific Aims1. Generation of muscle-specific creatine kinase driven Cre to generate muscle specific KO mice;and analysis of the skeletal and cardiac muscle development and function.2. Generation of NRIP transgenic mice and examination the effect on skeletal and cardiacmuscle development.3. Microarray analysis of NRIP-regulated myosin-related gene expression profile from wt andNRIP KO muscles.4. To generate R26R/NRIP-CreERT2 transgenic mice containing NRIP promoter drivendsRED-IRES-CreERT2 for genetic lineage tracing studies of the function of NRIP-expressingcells.5. Rescue effect of NRIP KO mice by AAV-8 promoter (skeletal and heart muscle specificpromoter) driven NRIP gene which is expressed in skeletal and cardio muscles by musclecontraction assays, Echocardiography and pathogenesis analysis.