內質網蛋白TXNDC5（硫氧還蛋白5）在心臟纖維化與心臟衰竭的角色

The Role of ER-Resident Protein TXNDC5 in Cardiac Fibrosis and Heart failure

陳昭伶(Chao-Ling Chen)

指導教授 ： 楊鎧鍵

國立臺灣大學/醫學院/藥理學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201602844

摘要

心臟衰竭 (heart failure) 是心臟因為先天異常或後天受損傷造成結構或功能異常，使得心臟搏出量不足以支應身體需求而產生症狀。心臟纖維化（cardiac fibrosis）在心臟衰竭病態之組織重組(remodeling) 扮演重要的一環。心臟纖維化的細胞病理特徵伴隨著心臟纖維母細胞被活化、其活化之分化態肌纖維母細胞(myofibroblast) 形成過程中促使細胞外組織間質膠元蛋白 （collagen）合成與沉積。心臟纖維化過程中產生大量的細胞外組織間質 (extracellular matrix) 堆積進而取代損壞心肌細胞，導致心室舒張功能異常、心肌電氣傳導受阻，增加嚴重心律不整發生的危險性。因此，針對心肌纖維化的治療可以減緩或防止心臟衰竭惡化。然而，目前可選擇的治療藥物相當有限，因此本研究希望找到與新穎的心臟纖維化分子機轉。本研究利用次世代高速核酸定序（Next-generation sequencing）針對人體左心室組織的RNA 表現進行分析，我們發現內質網蛋白TXNDC5 (Thioredoxin domain containing 5, 硫氧還蛋白5) 在心臟纖維化中扮演一個重要的樞紐基因。在人類與小鼠心臟衰竭組織與正常組織相較之下，TXNDC5 在心衰竭組織中具高度表現，其與纖維化相關基因Col1A1, ELN, CTGF 與ACTA2 表現呈現高度相關性，此外，TXNDC5 在小鼠心室也具有組織特異性，主要表現在心室纖維母細胞(ventricular cardiac fibroblasts)。然而，目前TXNDC5 在心臟纖維化過程中的機轉仍然不清楚。
因此我們的研究目標想要探討 (1) TXNDC5 參與心臟纖維化過程中的機轉，包括心臟纖維母細胞活性及基因/蛋白表現的分子機轉；(2) 並在動物模式中探討TXNDC5 在心臟纖維化的功能；(3) 以及未來針對TXNDC5 發展治療心臟纖維化藥物的可能性。
TXNDC5 已知為位於內質網具有雙硫鍵異構酶 (protein disulfide isomerase,PDI)，我們發現TXNDC5 於人類與小鼠的心室纖維母細胞在接受促進纖維化因子如 乙型腫瘤生長激素（tumor growth factor-β1, TGF-β1）刺激時，其表現量與心臟衰竭時表現相似。敲弱 (knockdown) 人類與小鼠心室纖維母細胞的TXNDC5 基因之後，我們發現其會減少為TGF-β1 刺激引起纖維化相關蛋白之製造與摺疊(folding)，但卻對纖維化相關基因表現沒有影響。另外我們也發現TXNDC5 基因敲弱的人類與小鼠心室纖維母細胞會透過內質網相關蛋白質降解路徑(endoplasmic reticulum associated protein degradation, ERAD) 的蛋白體酶(proteasome) 來降解未折疊之纖維化蛋白。而且TXNDC5 基因的表現為透過TGF-β1 引起內質網壓力 (ER stress) 下游所調控，其會受到ATF6 轉錄因子（Activating transcription factor 6）的調節，進而增加心臟纖維化蛋白質的疊疊與製造。在小鼠動物模型中，我們發現Txndc5-/-小鼠會減少因病理性刺激 （isoproterenol）導致的心臟肥厚、心肌纖維化以及心臟衰竭，改善左心室射出分率 (left ventricular ejection fraction)，並減少與纖維化相關蛋白質Col1A1, ELN, CTGF 與ACTA2 的表現。
因此本研究發現心臟組織/細胞受到病理壓力或TGF-β1 刺激，誘發內質網壓力調控下游的轉錄因子活化TXNDC5， TXNDC5 會藉由調控細胞外間質蛋白的折疊與蛋白質降解路徑，影響左心室纖維母細胞的活性與細胞外間質蛋白質的製造，造成心臟纖維化，為程中重要的調節因子。未來我們將設計可專一針對TXNDC5蛋白結合的核酸治療藥物DNA aptamer，發展出治療心臟纖維化及心衰竭的新治療模式與標的。

關鍵字

心臟纖維化 ； 心臟衰竭 ； TXNDC5（硫氧還蛋白5) ； 內質網壓力

並列摘要

Background：Heart failure (HF), one of the leading causes of increased morbidity, mortality, and healthcare burden, attributed largely to cardiac structural or function abnormalities in response to cardiac injury. Fibrosis plays a pivotal role in pathological remodeling of end-stage organ impairment. Cardiac fibrosis is characterized by the hyperproliferation of cardiac fibroblasts (CF), differentiation of CF into myofibroblasts, and excessive extracellular matrix (ECM) synthesis and deposition. Cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current diagnostic and treatment options for cardiac fibrosis, however, are very limited. Combining RNA sequencing in human failing heart, Weighted Gene Co-expression Network Analysis (WGCNA) and in vitro experiments, we have identified thioredoxin domain containing 5 (TXNDC5), an ER-resident protein, as a potential novel modulator of cardiac fibrosis. TXNDC5 is upregulated in failing human and mouse heart and its expression is highly correlated with the expression levels of fibrosis/ ECM genes including Col1A1, ELN, CTGF and ACTA2. However, it remains unclear how TXNDC5 regulation contributes to cardiovascular pathology such as cardiac fibrosis and heart failure.

Aim: The goal of this study was to investigate: (1) the molecular mechanisms of TXNDC5 modulated cardiac fibrosis; (2) in vivo functional contribution of TXNDC5 to cardiac fibrosis; (3) the therapeutic potential of targeting Txndc5 in preventing or reversing cardiac fibrosis.

Results: The ER-resident protein TXNDC5 is highly upregulated in human and mouse failing myocardium and in fibroblasts upon TGF-β1 stimulation. Knocking down TXNDC5 in cardiac fibroblasts abrogates TGF-β1-induced fibroblast activation, and ECM protein but not mRNA upregulation. Further experiments confirmed that TXNDC5 functions by facilitating the folding of ECM proteins; depletion of TXNDC5 leads to ECM protein misfolding and degradation through ER-associated degradation (ERAD) pathway. TGF-β1-induced TXNDC5 expression is dependent on transcriptional regulation downstream of ER stress pathway. To investigate the in vivo functional role of TXNDC5, we have generated Txndc5 KO (Txndc5-/-) mice using CRISPR/Cas9 genome editing technology. Comparing to WT animals, Txndc5-/- mice have reduced cardiac fibrosis/hypertrophy and preserved cardiac function in response to isoproterenol-induced cardiac injury. Furthermore, we tested the hypothesis that pharmacological inhibition of TXNDC5 using disulfide isomerase inhibitor 16F16 and DNA aptamers could be a novel therapeutic approach against cardiac fibrosis and heart failure.

Conclusion: We have identified an ER resident protein TXNDC5 as a novel mediator of cardiac fibrosis; Txndc5 regulates fibroblast activity and ECM production by modulating ECM protein folding and ER-associated protein degradation. TGF-β1, a critical mediator of cardiac fibrosis, triggers TXNDC5 expression in an ER stress-dependent manner. Genetic deletion of TXNDC5 protects against β agonist-induced cardiac fibrosis and LV dysfunction. Therefore, TXNDC5 represents a novel molecular mechanism contributing to cardiac fibrosis and could be a novel therapeutic target against cardiac fibrosis and heart failure.

並列關鍵字

cardiac fibrosis ； thioredoxin domain containing 5,TXNDC5 ； endoplasmic reticulum stress

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