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

Abstract

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。

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.