- 學位論文
肢帶型肌肉失養症所引起之心臟功能異常與NRIP(細胞核受體交互作用蛋白質)異常調控的關係
Dysregulation of Nuclear Receptor Interaction Protein (NRIP) Contributes to Cardiac Dysfunction Associated with Limb-Girdle Muscular Dystrophy
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摘要
肢帶型肌肉失養症 (Limb Girdle Muscular Dystrophy, LGMD)是一群退化性肌肉疾病的統稱，通常會有四肢骨骼肌無力的情形，依據不同的疾病亞型影響不同的肌肉組織。在心臟部分，此類型的肌肉失養症可能會造成收縮異常抑或是心源性猝死(sudden cardiac death)。然而經由LGMD所引起心肌異常的分子機轉並不為人所知。細胞核受體交互作用蛋白質(Nuclear Receptor Interaction Protein, NRIP, Dcaf6, IQWD1)是一個鈣離子依賴型的鈣調蛋白結合蛋白(calcium-dependent calmodulin-binding protein)，在LGMD患者的骨骼肌當中表現量下降；而在NRIP基因剃除小鼠(Nrip-/-)當中，也發現他們有和LGMD患者相同的肌肉功能低落(muscle weakness)的現象。然而，目前LGMD所引起的心臟功能異常以及其和NRIP的關係機轉並不清楚。 因此，本研究利用NRIP橫紋肌專一性基因剔除小鼠(MCK-Cretg/+; Nripfl/fx)針對橫紋肌如骨骼肌、心肌等做基因剔除，想要探討：(1) NRIP缺失對於心臟收縮功能的影響以及之間的分子機轉；(2) 未來NRIP是否能作為治療LGMD所引起之心臟疾病的目標基因。 NRIP在橫紋肌的專一性剔除曾被報導和骨骼肌的收縮功能有密切相關，在我們的研究中，發現NRIP對於心臟亦有相似的影響，MCK-Cretg/+; Nripfl/fx相對於正常的wild-type老鼠心臟收縮功能較差(左心室射血分數[LVEF] 43.447 ± 1.660% vs 57.844 ± 5.344% in MCK-Cretg/+; Nripfl/fx and WT, respectively, P<0.01.) 測量自MCK-Cretg/+; Nripfl/fx左心室分離心肌細胞之收縮強度(contractility)、鈣離子流動速率(calcium transient)以及肌漿網內含鈣量(sarcoplasmic reticulum calcium store)和wild-type小鼠之細胞相比都有明顯的下降。然而，我們利用次世代高核酸定序(Next generation sequencing)發現調控鈣離子之相關基因，如：L-type Ca2+ channel以及SERCA2a，在NRIP缺失與WT之間的表現量並沒有顯著差異。由於心臟收縮功能與ATP的產生有密切相關，又粒線體為細胞中主要的能量產生胞器，我們利用穿透式電子顯微鏡的技術，觀察心肌上的粒線體，發現NRIP缺失小鼠有粒線體結構上的異常，尤以cristae密度下降最為明顯；我們研究也發現，粒線體中的活性氧物種(Reactive oxygen species, ROS)在NRIP缺失時上升且NAD+/NADH 比例下降。在HL1心肌細胞中敲弱 (knockdown) NRIP亦會導致粒線體中的ROS增加、呼吸鏈功能異常且ATP產量下降；MCK-Cretg/+; Nripfl/fx的心肌細胞當中也有相同的結果。此外，我們研究也指出MCK-Cretg/+; Nripfl/fx會影響脂肪酸氧化(fatty acid oxidation)相關基因，並且使得脂肪酸氧化作用增加，導致NAD+及NADH含量失衡。我們也發現，在MCK-Cretg/+; Nripfl/fx小鼠中給予粒線體ROS抑制劑－mitoTEMPO可減少粒線體中的ROS，且有效的回復心臟收縮功能(左心室射血分數[LVEF] 50.299 ± 0.704 % vs 44.362 ± 1.903% in mitoTEMPO treated MCK-Cretg/+; Nripfl/fx and phosphate buffer saline [PBS] treated MCK-Cretg/+; Nripfl/fx, respectively, P<0.05)。另外，利用NAD+的前驅物－菸鹼酸(nicotinic acid)亦能有效的在MCK-Cretg/+; Nripfl/fx小鼠中提升其心臟功能(左心室射血分數 [LVEF] 55.887 ± 1.981 % vs 45.227 ± 1.672 % in nicotinic acid treated MCK-Cretg/+; Nripfl/fx and phosphate buffer saline [PBS] treated MCK-Cretg/+; Nripfl/fx, respectively, P<0.05)。 因此本研究發現NRIP缺失會導致粒線體cristae密度下降、粒線體中的ROS上升，造成粒線體氧化壓力上升、呼吸鏈功能異常、ATP產量下降，最終使得心臟收縮能力下降。我們的結果推測粒線體的ROS上升是由於NAD+/NADH比例下降，且造成此比例下降的主要原因為脂肪酸氧化過程增加，使NAD+、NADH含量失衡，最終造成粒線體氧化壓力上升、ATP產量下降，導致心臟功能受損。針對NRIP在心臟方面的相關研究，對於LGMD所引起的心臟功能異常病人能提供一個新的治療標的。
並列摘要
Background: Limb-girdle muscular dystrophy (LGMD), a muscular dystrophy that predominantly affects proximal limb muscles, frequently involves the heart, leading to contractile dysfunction or sudden cardiac death. The molecular basis underlying LGMD-associated myocardial dysfunction, however, remains elusive. Nuclear receptor interaction protein (NRIP), a Ca2+-dependent calmodulin-binding protein also known as DCAF6 or IQWD1, is downregulated in the skeletal muscles of patients with LGMD. Mice with targeted deletion of NRIP (Nrip-/-) were found to have muscle weakness that mimics LGMD. We sought to test mechanistically the hypothesis that reduced NRIP expression could contribute to cardiac dysfunction observed with LGMD. Purpose: To investigate the molecular mechanisms via which NRIP deficiency contributes to cardiac dysfunction and to further explore potential therapeutic approach against NRIP deficiency-induced cardiac dysfunction and LGMD-related cardiomyopathy. Methods and Results: Striated muscle-specific Nrip knockout mice (MCK-Cretg/+; Nripfl/fx) were found to have markedly reduced cardiac contractile function, compared to WT control (left ventricular ejection fraction [LVEF] 43.447 ± 1.660% vs 57.844 ± 5.344 % in MCK-Cretg/+; Nripfl/fx and WT, respectively, P<0.01). Isolated MCK-Cretg/+; Nripfl/fx LV myocytes showed significantly reduced contractility, peak Ca2+ transient amplitudes, and rate of Ca2+ transient decay, compared with WT LV cells. RNA sequencing did not reveal significant differences in the expression levels of Ca2+ handling proteins such as L-type Ca2+ channels and SERCA2a, between MCK-Cretg/+; Nripfl/fx and WT LV. Electron microscopy discovered changes in mitochondrial morphology with reduced cristae density in MCK-Cretg/+; Nripfl/fx LV, which was accompanied with increased mitochondrial ROS levels and reduced NAD+/NADH ratio. In addition, fatty acid oxidation associated genes were increased in MCK-Cretg/+; Nripfl/fx mice, which may result in an imbalance of the amount of NADH and NAD+. Knocking down NRIP in mouse HL1 cardiomyocytes led to increased mitochondrial ROS, impaired mitochondrial respiratory function and reduced ATP production, which were also observed in isolated LV myocytes from MCK-Cretg/+; Nripfl/fx mice. Treatment with mitochondria-directed antioxidant mitoTEMPO significantly attenuated the contractile dysfunction observed in MCK-Cretg/+; Nripfl/fx mice (left ventricular ejection fraction [LVEF] 50.299 ± 0.704 % vs 44.362 ± 1.903% in mitoTEMPO treated MCK-Cretg/+; Nripfl/fx and phosphate buffer saline [PBS] treated MCK-Cretg/+; Nripfl/fx, respectively, P<0.05). Treatment with nicotinic acid, a NAD+ precursor, also significantly enhanced the contractile function observed in MCK-Cretg/+; Nripfl/fx mice (left ventricular ejection fraction [LVEF] 55.887 ± 1.981 % vs 45.227 ± 1.672 % in nicotinic acid treated MCK-Cretg/+; Nripfl/fx and phosphate buffer saline [PBS] treated MCK-Cretg/+; Nripfl/fx mice, respectively, P<0.05) through reversed the imbalance of NADH and NAD+. Conclusion: NRIP depletion leads to impaired cardiac contractility, Ca2+ homeostasis and mitochondrial function as a result of increased mitochondrial oxidative stress secondary to aberrant NAD+/NADH ratio which was caused by the increment of fatty acid oxidation associated genes. Targeting NRIP and mitochondrial oxidative stress could be a potential therapeutic approach to treat or prevent cardiac dysfunction associated with LGMD.
參考文獻
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延伸閱讀
- Chen, C. H. (2014). 第二型富含半胱胺酸蛋白質在血管平滑肌細胞表現之調節機制以及其調控細胞移動之機轉 [doctoral dissertation, National Tsing Hua University]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0016-2912201413540188
- Wang, Y. S. (2022). NRIP自體抗體在肌萎縮性脊髓側索硬化症所扮演的角色 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU202201959
- Lu, Y. Y. (2014). 細胞外間質與整合素交互作用於心臟纖維化過程中對於心房及肺靜脈心肌細胞電生理重塑機轉之研究 [doctoral dissertation, Taipei Medical University]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0007-1102201413595100
- Wu, T. C. (2016). Muscle-derived NRIP deficiency causes age-dependent progressive motor neuron degeneration and motor deficits [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU201603184
- Lin, H. C. (2016). The Hypoxic Responsive NDRG-OT1_v4 Promotes NDRG1 Degradation via Ubiquitin-Mediated Proteolysis in Breast Cancer MCF-7 Cells [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU201601324