內質網蛋白TXNDC5 (硫氧還蛋白5) 經由促進細胞外基質的蛋白質折疊及活化氧化還原敏感性心室纖維母細胞以增強心臟纖維化心臟纖維化的惡化

Abstract

心衰竭目前是主要且已經形成全球矚目的公共健康問題,全球至今已有兩千三百萬的人患有心衰竭，且患有心衰竭的病患其五年內的死亡率仍維持在50%,造成心衰竭的最主要因素為心纖維化,其病理特徵為有過多的細胞外基質堆積而造成心室壁變得堅硬,導致心臟的結構重塑, 心功能異常,心臟電氣傳導失常導致心律不整...等致死之危險因子。使用RAAS拮抗劑, angiotensin II receptor及aldosterone receptor阻斷劑雖然可改善心室功能及減緩心臟纖維化,但其 會造成低血壓的副作用,並且未能有效阻止心臟纖維化的現象。發炎因子TNF-α也被認為是造 成心臟纖維化的原因之一,但使用TNF-α拮抗劑- etanercept及infliximab不僅分別會造成心衰竭 病患出現負面影響,其甚至還會增加中重度慢性心衰竭患者的死亡率。另外，雖然使用 endothelin抑制劑在各種器官纖維化的實驗模型顯示有降低纖維化的效果,可惜的是,其會造成嚴重的液體滯留在四肢導致水腫。臨床上使用廣效型TGFβ抑制劑, pirfenidone及tranilast,雖然報導指出其可以有效抑制纖維母細胞活化以及細胞外基質的沉積,但使用此類藥物卻會產生肝毒性。再者,雖然有許多研究發現和心纖維化有關的新分子,像是非編碼核糖核酸miR-21, miR-29, lncRNA,但在臨床的實際應用技術上仍處於未成熟期。即使有許多研究有利用哺乳類動物模型 為基礎進行試驗,且實驗結果非常具有希望,但其數據及樣本量低,仍然不足以驗證確實可使用 於人體中,即使進到臨床試驗,結果卻往往不如預期。因此目前找到具有潛力專一性治療心纖維 化的分子並且確切揭開其致病機轉有著迫切的需求。我們收集了心纖維化病患的左心室,並萃 取其RNA,進行RNA sequencing,利用co-expression network analysis及gene ontologic analysis找到了1071個genes和collagen fibril organization及extracellular matrix organization有相關,其中有一個 基因為TXNDC5,其與纖維化的基因像是fibronectin, collagen, elastin, TGFβ1有高度正相關。 敲弱 (knockdown) 人類與小鼠心室纖維母細胞的 TXNDC5/Txndc5 基因之後，我們發現其會減少被TGFβ1刺激所引起纖維化相關蛋白的製造，但卻對纖維化相關基因表現沒有影響。因 此我們利用protein degradation assay來測定在不同時間點的纖維化蛋白降解速率,並且發現 TXNDC5的敲弱可增加未成熟的纖維化蛋白的降解速率,並且這樣的降解路徑可能是藉由 ER- associated degradation pathway (ERAD)。此外,我們也發現ERAD路徑中有一種 ATP 水解酶複 合物稱作VCP (Valosin Containing Protein), 其會將未折疊好的蛋白拉出 ER 外, VCP 會供給 ERAD中的其他蛋白質ATP, 此能量會促進未折疊好的蛋白質被其他ERAD factors進行一連串 降解級連效應(cascade)。為了證實我們假說, 我們在敲弱人類纖維母細胞的TXNDC5基因後給 予ERAD inhibitor: Eeyarestatin I,發現此藥物可以反轉纖維化蛋白降解的現象;同時敲弱人類纖 維母細胞的TXNDC5基因及VCP基因也會得到相同的效果。另外,使用FRET (fluorescence resonance energy transfer)-based protein folding assay,發現在敲弱(knockdown)人類與小鼠心室纖 維母細胞的TXNDC5/Txndc5基因之後所偵測到的Col1A1/ELN FRET signal efficiency也有顯著 下降,說明著TXNDC5的功能可能確實會藉由折疊組織間質蛋白質來使此類型的蛋白質成熟度 更為完整。 在人類及老鼠纖維母細胞中TXNDC5基因會因為受到TGFβ1刺激而誘發內質網壓力,因而活 化ATF6轉錄因子進入細胞核,進而來調節TXNDC5/Txndc5基因表現,進而增強心臟纖維化蛋白質的折疊與製造,除此之外, 活化後的TXNDC5可能還會活化NADPH oxidase 4 來增加活性氧化 物(Reactive oxygen species),進而活化p-JNK,使得纖維母細胞轉型成肌纖維母細胞,這樣一連串的級連效應(cascade)會造成人類心肌纖維母細胞的累積,而形成不可逆性的心臟功能衰竭疾病。 為了進一步了解TXNDC5促進細胞外基質蛋白的折疊以及活化NADPH oxidase 4的作用是否 與其protein disulfide isomerase的功能有關,我們於TXNDC5上的三個進行protein disulfide isomerase作用的thioredoxin domain進行點突變,並將其表達於人類纖維母細胞中,發現突變後的 thioredoxin domain可造成TXNDC5的功能喪失,並且減少人類纖維母細胞ROS製造,也降低各種纖維化相關蛋白質 Col1A1, ELN, CCN2的表現,同時甚至減緩人類纖維母細胞的活化及轉型,由此可知, thioredoxin domain對於TXNDC5 促進心臟纖維化有重要的角色。 於 in vivo 的部分,我們使用了isoproterenol引致心衰竭的小鼠動物模型,並以M-mode echocardiographic images分析小鼠的心臟功能,結果發現Txndc5基因剔除 (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,進而改善左心室射 出分率(left ventricular ejection fraction)。並且isoproterenol刺激的Txndc5-/-小鼠相較於 Txndc5+/+小鼠,存活率也有明顯上升,由此可說明在心臟受到刺激或壓力下, Txndc5 基因剔除 具有保護心臟的效果。 在本篇研究中,我們對於 TXNDC5 的功能有重大的發現,其會透過兩種機轉來造成心臟纖維化,一為促進細胞外間質蛋白的折疊,二為活化NOX4來增加心臟纖維母細胞的氧化壓力,進而增加 JNK 的磷酸化,進而促進心臟纖維母細胞的增生,本研究提供了一個針對心肌纖維化與心臟衰竭極具潛力的治療策略,也期待未來能夠作為治療纖維化的新藥開發分子。

Background: Heart failure (HF) is a global pandemic health issue, afflicting more than 26 million people worldwide and is still rising in prevalence. HF carries significant 5-year mortality, at the forefront of those many cancers. Besides, HF staggering annual global expenditure on healthcare systems. Myocardial fibrosis is strongly the pivotal role in pathological remodeling of end-stage organ impairment such as facilitates the development of arrhythmogenicity, cardiac dysfunction, also impacts the clinical course and outcome in patients with heart failure. Cardiac fibrosis is featured as long-term cardiac fibroblasts (CF) persistent proliferation, differentiation of CF into myofibroblasts, and extreme extracellular matrix (ECM) secretion and deposition. Meanwhile, fibrosis impairs cardiac contractile function and increases the risk of sudden death. Antagonists of RAAS, such as angiotensin converting enzyme inhibitor, angiotensin II receptor and aldosterone receptor blockers, have been shown to improve ventricular function and slow progression of myocardial fibrosis. The use of these treatments, however, is limited by their hypotensive effects and their inability to stop fibrosis progression. Inflammation modulators, TNF-α plays an important role in cardiac fibrosis. TNF-α antagonist- etanercept and infliximab, nevertheless, has negative effect in heart failure patients and increase moderate-to-severe chronic heart failure patient’s all-cause mortality independently. Endothelin inhibitor, in spite of that it prevents several organ fibrosis in animal models, anyhow, it will mostly enhance fluid retention. Broad inhibitors of TGFβ such as pirfenidone and tranilast, on the other hand, have been shown to attenuate fibrosis by inhibiting fibroblast activation and ECM deposition without affecting blood pressure in experimental models. The widespread clinical use of these agents, however, is imperfect due to undesirable side effects, including liver toxicity. On the other hand, although several researches has identified new molecules of cardiac fibrosis such as miR- 21, miR-29, lncRNA. The challenges that techniques for ncRNA­based therapies are remaining infancy. Even though various experiment has been assay under the preliminary mammal groundwork, and seem promising, the data are even now limited and mixed. Numerous budding novel treatments which have displayed to lessen cardiac fibrosis in animal models have either not been verified in humans or shown to be disappointing in clinical trials. Consequently, uncover and clarify additional mediators of cardiac fibrosis turn out to be crucial to expedite the advance of new therapeutic approaches targeting myocardial fibrosis. In this study, manipulating RNA sequencing in human failing heart, Weighted Gene Co-expression Network Analysis (WGCNA) displays that thioredoxin domain containing 5 (TXNDC5), an ER chaperone protein, as a potential innovative modulator of cardiac fibrosis. TXNDC5 is upregulated in failing human and mouse heart and its expression is vastly correlated with the expression levels of fibrosis/ECM genes including Col1A1, ELN, CTGF and ACTA2. However, it remains to shed light on how TXNDC5 trigger the myocardiac fibroblasts activation that contributes to cardiovascular pathology such as cardiac fibrosis and heart failure. Aim: Exploring the comprehensive molecules and pathways involved in: (1) the 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: Knocking down TXNDC5 in cardiac fibroblasts abolishes TGF-β1-induced fibroblast activation, and ECM proteins upregulation independent of SMAD3. More investigations report that TXNDC5 performs as post-translational modification by expediting the folding of non-native proteins; indeed, depletion of TXNDC5 brings about ECM proteins misfolding but not mRNA upregulation, and lead to further proteins degradation through ER-associated degradation (ERAD) pathway. We also found out TGF-β1-induced TXNDC5 expression is reliant on ER stress pathway downstream factor activating transcription factor 6(ATF6) transcriptional regulation. TXNDC5 promotes hCF activation and proliferation by enhancing JNK activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. To clearify the in vivo role of TXNDC5, we generated CRISPR/Cas9 genome editing Txndc5 knockout (Txndc5-/-) mice. Comparing to WT animals, Txndc5-/- mice reduced cardiac fibrosis/hypertrophy and preserved cardiac function in response to isoproterenol-induced cardiac injury. Conclusion: We have recognized an ER resident protein TXNDC5 as a potential mediator of cardiac fibrosis; TXNDC5 promotes fibroblast awake and ECM production by modulating ECM protein folding and redox-sensitive JNK signaling. TGF-β1 play a perilous mediator of cardiac fibrosis, facilitates TXNDC5 expression in an ER stress-dependent approach. Genetic loss of TXNDC5 mitigates against β agonist-induced cardiac fibrosis and LV dysfunction. Therefore, TXNDC5 represents a novel therapeutic approach to improve cardiac function and outcomes in HF patients.