利用腺相關病毒(AAV)進行因缺失核受體結合蛋白(NRIP)所造成心臟不正常之基因治療

Abstract

核受體結合蛋白 (Nuclear receptor interaction protein, NRIP) 又名DCAF6或IQWD1，此蛋白質帶有七的WD-40 repeats以及一個IQ motif，且當有鈣離子的情況下，NRIP會以IQ motif與攜鈣素 (calmodulin; CaM) 結合，進而調控與肌肉功能相關之下游鈣離子信息，在本實驗室所建立之肌肉NRIP基因剃除小鼠(muscle-restricted NRIP knockout mice, cKO mice)上也看到肌肉以及運動功能異常的情況。NRIP同時為一個肌小節Z盤 (Z-disc) 蛋白，先前研究發現NRIP能以IQ motif和主要構成Z-disc的輔肌動蛋白 (α-actinin2; ACTN2) 結合，且NRIP除了影響骨骼肌外，在肌肉NRIP基因剃除小鼠 (NRIP-cKO) 也發現有心臟收缩力下降,心肌肥大 (hypertrophy) 鈣離子流動速率 (calcium transient) 下降，以及粒線體呼吸功能和型態不正常的現象，因此初步發現認為NRIP可能扮演維持心臟肌肉正常功能之角色。 首先我們發現12周之NRIP-cKO老鼠相對於正常的wild-type (WT) 老鼠有心臟收收功能下降的情況；另外我們也利用穿透式電子顯微鏡 (Transmission Electron Microscopy; TEM) 觀察其心肌肌節型態，進一步看到cKO老鼠肌節上的Z-disc變寬以及I-band變短的現象，與先前的研究結果一致；加上我們進一步在年紀較大 (22 周) 的cKO老鼠身上發現有心肌細胞 (cardiomyocyte) 變大的情形。因此，我們認為NRIP在心肌上扮演穩定Z-disc結構的角色，NRIP的缺失可能造成肌節結構的不正常以及心臟整體收縮功能下降，並引發漸進的心臟肥大 (cardiac hypertrophy). 然而，關於NRIP缺失如何影響心臟功能及型態的機轉並不清楚，因此本研究的第一個目標為探討NRIP在心肌上的分子機轉。由於NRIP能與ACTN2結合，而ACTN2是構成Z-disc 的主要蛋白，能與許多其他Z-disc交互作用及聯結纖維狀肌動蛋白 (F-actin)，我們發現NRIP-cKO老鼠心臟之ACTN2與Cap-Z結合能力較差，此結果顯示NRIP對於維持ACTN2與Cap-Z結合的穩定性是重要的。除此之外我們也利用in vitro sedimentation assay，發現NRIP會影響ACTN2與F-actin的親和力。總結來說，我們的研究結果顯示NRIP在Z-disc上可能透過影響actin/ACTN2以及其他相關蛋白來調控穩定心肌結構的角色。 根據目前結果顯示NRIP的缺失會造心臟收縮功能下降，Z, I-band的不正常以及在分子機轉上降低ACTN2與Cap-Z結合的穩定性；因此在本研究的第二個部分，我們利用帶有NRIP基因之腺相關病毒(AAV)進行基因療法，欲達到治療NRIP缺失所導致的不正常現象。然而，利用心包內注射 (intrapericardial injection) 給予出生後五天老鼠 AAV的治療之後，我們在12周進行心臟超音波(Echocardiography)診斷心臟收縮功能是否改進，心肌肌節型態是否恢復以及其他相關觀察，發現在NRIP-cKO老鼠上給予AAV的基因治療無法有效改善因缺失NRIP所導致之現象，我們初步認為此結果的可能原因為AAV在心臟內的表現程度不足無法有效補足NRIP表現量所造成。 最後，除了上述所提NRIP扮演穩定肌節的功能以外，我們也探討NRIP的其他分子機轉。由於NRIP能以其IQ motif與ACTN2和CaM結合；根據其他研究指出許多同樣能和ACTN2和CaM結合的蛋白能透過鈣離子 (Ca2+) 來調控蛋白在細胞膜上的表現或離子通道的活性，因此我們想探討Ca2+對於 NRIP-ACTN2-CaM三者之間交互作用的影響。利用在293T細胞的免疫沉澱法(immunoprecipitation) 初步結果發現，NRIP-ACTN2-CaM能形成附合體，然而當Ca2+濃度增加時，NRIP會傾向與CaM結合，使得NRIP與ACTN2之間的親合力降低。除此之外，我們也發現NRIP除了透過ACTN2影響F-actin的連結 (cross-link) 以外，本身能直接或接間接與actin交互作用；此結果也暗示NRIP可能參與其他在細胞骨架或細胞膜上的分子作用機轉。

Nuclear receptor interaction protein (NRIP, also known as DCAF6 and IQWD1) consists of 860 amino acids and encodes seven WD40 domains with one IQ motif. NRIP interacts with calmodulin (CaM) in the presence of calcium via the IQ domain. NRIP has been reported to be a Z-disc protein, and NRIP-knockout mice were found to have reduced muscle strength, and impaired regenerative capacity in skeletal muscle. In addition, our unpublished results revealed that muscle-specific NRIP knockout mice (NRIP-cKO) displayed progressive cardiac hypertrophy along with impair contractile function and decrease of calcium transient as well as abnormal mitochondrial morphology along with reduced mitochondrial respiration efficiency. And NRIP could bind the EF hand of actinin2 (ACTN2) by its IQ motif, ACTN2 is a critical Z-disc component crossing actin filament. Taken together, all these results suggest that NRIP may play an important role in heart. In this study, we extensively investigated NRIP role in heart functions. Firstly, we found the impaired cardiac function in 12 week NRIP-cKO mice compared with WT. Ultrastructural analysis by transmission electron microscopy (TEM) further reveled the Z-disc widening and I-band reduction in cKO mice compared with WT, which are consist with the previous observations in global NRIP knock out mice. In addition, we found the enlargement of cardiomyocyte size in NRIP-cKO mice at the age of 22 weeks but not 12 weeks. According to the functional and structural abnormalities in cKO mice, we assume that NRIP might be essential for Z-disc ultrastructure supporting, loss of NRIP in heart causes ultrastructural changes and therefore leads to cell size enlargement and progressive hypertrophy. Next, we surveyed the molecular mechanism that contributes to the cardiac structural and contractile abnormalities caused by NRIP deficiency. As NRIP is the binding protein of ACTN2, we searched several ACTN2 binding partners, and found the binding affinity between ACTN2 and Cap-Z significantly reduced in NRIP-cKO mice compared with WT, which indicated that NRIP plays an important role in stabilizing the binding between ACTN2 and Cap-Z at Z-disc. Apart from NRIP role in ACTN2-Cap-Z binding stabilization, we were also interested in whether NRIP has potential to promote α-actinin–actin binding affinity. By in vitro sedimentation assay, it showed that NRIP was able to enhance the cross-link of F-actin and ACTN2. In conclusion, we demonstrated that NRIP serves as a scaffold protein which bind the EF hand of ACTN2 and stabilizes the binding between ACTN2-Cap-Z as well as ACTN2 to cross-link F-action. In cardiomyocytes, NRIP might cooperate with ACTN2 to enhance the stability or mechanical strength of the sarcomere. Considering NRIP deficiency leads to impaired cardiac function, abnormality of Z and I-band length and affects ACTN2-Cap-Z binding affinity as described above. We next delivered AAV-NRIP through intrapericardial injection in neonatal mice and attempted to rescue the phenotypes seen in NRIP-cKO mice. The mosaic distribution of NRIP protein expression could be detected at age 22 weeks after AAV injection in hearts. However, no significant recovery of cKO mice with AAV-NRIP treatment was observed compared with AAV-GFP treatment by analyzing several parameters, including cardiac function, TEM ultrastructure, cell size and ACTN2-Cap-Z binding affinity, which might be explained by low AAV infection efficiency. In the last part of this study, we searched several novel characteristics of NRIP in addition to its role in Z-disc structure integrity. Based on the previous reports, NRIP can interact with ACTN2 and CaM through its IQ motif. Other IQ containing proteins such as N-Methyl-D-aspartate receptors (NMDAR) and L-type Ca2+ channels are reported to interact with both CaM and ACTN2, in the present of Ca2+ , the interactions of CaM and ACTN2 with these protein have affected on either ion-channel activity or protein expression on cell membrane. Thus, we further investigated the interaction among NRIP, ACTN2 and CaM with or without Ca2+, the results showed that these three proteins were able to form a stable complex under normal condition, while when the Ca2+ level was raised, NRIP could bind CaM with a higher affinity than ACTN2. In addition, by immunoprecipitation assay, the evidence suggested that NRIP might directly or indirectly bind to F-actin in ACTN2-independent manner. Collectively, these results imply the potential of NRIP to regulate the cytoskeletal integrity and molecular mechanisms on cell membrane in addition to maintaining Z-disc structure stabilization.