第二型血管收縮素轉化酶缺乏與阿黴素誘發急性心肌病相關性之研究

Abstract

阿黴素（Doxorubicin；DOX）是一個被廣泛使用的抗癌藥物，但其具有致命的心毒性，會導致心肌細胞的死亡並造成心臟重塑、心臟功能受損及心臟衰竭。細胞外基質（Extracellular matrix; ECM）的不正常代謝已被發現在發展心臟衰竭的過程中會參與心臟組織結構的重塑。ECM的平衡和代謝受到基質金屬蛋白酶（Matrix metalloprotease; MMPs）及其內源性組織抑制因子（Tissue inhibitor of metalloproteinases; TIMPs）的嚴密調控，許多研究指出MMPs和TIMPs的失衡和不利的心臟組織重塑及心肌病的發展有很高的相關。腎素-血管收縮素系統（Renin-angiotensin system; RAS）是體內調控心臟、血管和腎臟的賀爾蒙系統，並且和心血管疾病的關係密切。第二型血管收縮素轉化酶（Angiotensin converting enzyme II; ACE2）是一能將第二型血管收縮素（Angiotensin II; Ang II）降解為血管收縮素1-7（Angiotensin 1-7; Ang 1-7）的酵素，ACE2對RAS具有負調控作用，能減低Ang II引發的發炎反應、纖維母細胞生長與分化、心肌細胞凋亡以及心臟組織纖維化，然而ACE2對心臟疾病的影響卻還有許多地方尚未釐清。 ACE2如何影響MMPs/TIMPs的平衡，以及這些影響是否受到心肌細胞的訊息傳遞路徑的調控是我們感興趣的議題。我們推測ACE2在誘發性的心肌病中具有保護的功能，我們假設（1）MMPs/TIMPs的失衡在缺乏ACE2的情況下將被增強，以及（2）在誘導的急性心肌病中，缺乏ACE2會使得細胞外訊息調控因子（Extracellular-signal-regulated kinase；ERK1/2）和訊息傳遞轉錄活化子（Activator of transcription 3; STAT-3）等訊息傳遞路徑被進一步的活化或是抑制，並經由RAS相關的因子以影響MMPs/TIMPs的平衡。 在本研究中，我們經由腹腔注射給予小鼠DOX，每次注射10 mg/kg，共分兩次，三天注射一次，以誘發小鼠的急性心肌病。為了瞭解ACE2的影響和其在心肌病中所扮演的角色，我們利用野生型（Wild type; WT）與ACE2基因剔除（ACE2 Knockout; ACE2 KO）小鼠進行實驗，並且比較在這兩種小鼠心臟組織中MMPs、TIMPs、pERK1/2和pSTAT3的表現樣態。所有實驗的小鼠皆於第一次注射給藥後的七天犧牲並且採集其心室樣本。我們作明膠酶譜（Gelatin zymography）分析MMP-2、MMP-9的活性，用西方墨點法（Western blot）測定TIMP-1、TIMP-2、pERK1/2及pSTAT3的表現量。 實驗結果顯示，DOX誘發的心肌病會造成小鼠體重顯著地下降和使血漿中肌酸激酶（Creatine kinase; CK）的活性顯著提升。在WT小鼠的心臟組織中，施予DOX造成MMP-2與MMP-9活性及pERK1/2與pSTAT3的表現量顯著的上升，TIMP-1與TIMP-2的表現量只有些微上升。而在ACE2 KO小鼠的心臟組織中，DOX造成MMP-9的活性和pERK1/2的表現量顯著上升，MMP-2的活性與pSTAT3的表現量也都有上升但並不顯著，而TIMP-1與TIMP-2的表現量則只有些微上升。比較結果顯示，DOX會造成小鼠心臟組織的MMP-2和MMP-9活性上升而且在ACE2 KO小鼠中， MMP-2和MMP-9的活性明顯低於WT小鼠，ACE2 KO小鼠的TIMP-1、TIMP-2與pSTAT3的表現量則稍微地低於WT小鼠，而pERK1/2的表現量則都有顯著提升但兩者間沒有明顯的差異。 統整以上實驗結果，我們發現在DOX誘發小鼠急性心肌病過程中，缺乏ACE2沒有導致MMPs的活性更進一步被提升，反而抑制了MMPs活性的提升，但缺乏ACE2對TIMPs和pERK2以及pSTAT3的表現影響甚微。儘管我們無法證明ACE2在心肌病過程中扮演的角色，本實驗結果仍然證明RAS相關分子在DOX誘發急性心肌病中可能影響MMPs和TIMPs分子的表現調節，而此可能與pERK1/2和pSTAT3的訊息傳遞路徑有關。

Doxorubicin (DOX) is the most widely used anticancer drug with lethal cardiotoxicity which causes death of cardiac myocytes and leads to heart remodeling, heart dysfunction and heart failure (HF). The abnormal metabolism of extracellular matrix (ECM) has been reported participates in structural remodeling of heart tissue in the development of HF. The balance of ECM is precisely modulated by matrix metalloproteinases (MMPs) and their internal inhibitors, tissue inhibitor of metalloproteinases (TIMPs). Many studies have shown that, imbalance of MMPs/TIMPs are highly associated with the adverse heart remodeling and the development of cardiomyopathy. Renin-angiotensin system (RAS) is a hormone system modulating heart, vessels, and kidney, and it is associated with the pathogenesis of cardiovascular diseases. Angiotensin converting enzyme II (ACE2) is an enzyme degrades angiotensin II (AngII) into angiotensin 1-7 (Ang 1-7), and acts as a negative regulator of RAS against Ang II-mediated inflammation, fibroblasts growth and differentiation, cardiac myocyte apoptosis, and heart tissue fibrogenesis,. However, the effects of ACE2 in heart disease are still not well described. It is interested how ACE2 involved in the imbalance of MMPs/TIMPs and whether the effects of ACE2 on MMPs/TIMPs is mediated by other signaling pathways. We assume that ACE2 has protective function in induced cardiomyopathy. We presume that (1) the imbalance of MMPs/TIMPs are enhanced when loss of ACE2, and (2) signaling pathways such as extracellular-signal-regulated kinase (ERK1/2) and activator of transcription 3 (STAT-3) could be activated or inhibited which further influence the balance of MMPs /TIMPs through mediators involved in RAS in induced acute cardiomyopathy when loss of ACE2. In this study, 10 mg/kg of DOX were intraperitoneal injected twice with three-day interval to induce acute cardiomyopathy in mice. To figure out the effects and the role of ACE2 in cardiomyopathy, both wild type (WT) mice and ACE2 knockout (ACE2 KO) mice were employed to compare the expression profiles of MMPs, TIMPs, pERK1/2 and pSTAT3. Mice were sacrificed and the ventricles of mice were harvested on the 7th day after the first injection. Gelatin zymography was utilized to determine the activity of MMP-2, MMP-9 while western blot was applied to determine the expression of TIMP-1, -2, pERK1/2 and pSTAT3. Our results show that DOX-induced cardiomyopathy results in marked decrease in mice body weight and dramatic increase in plasma creatine kinase (CK) activity. In WT mice ventricular tissue, with DOX treatment, the activity of MMP-2 and MMP-9 and the expression of pERK1/2 and pSTAT3 were obviously up-regulated while the expressions of TIMP-1 and TIMP-2 were slightly increased. In ACE2 KO mice ventricular tissue, DOX treatment leads to significant up-regulation of MMP-9 activity and pERK1/2 expression, increase, but not significant, in MMP-2 activity and pSTAT3 expression, and slightly higher expressions of TIMP-1 and TIMP-2. Compare those results, the DOX-induced increase in MMP-2 and MMP-9 activity in ACE2 KO mice ventricular tissue was obviously lower than WT. The expressions of TIMP-1, -2 and pSTAT3 in ACE2 KO mice ventricular tissue slightly lowered than WT mice ventricular tissue, and the expressions of pERK1/2 were both increased with no difference. Based on our results, we found that in the pathogenesis of DOX-induced acute cardiomyopathy in mice, loss of ACE2 does not lead to further activation of MMPs. On contrary, loss of ACE2 inhibits the upregulation of MMP activity without affecting the expression of TIMPs, pERK1/2 and pSTAT3. Although we can not prove the role played by ACE2 in pathogenesis of cardiomyopathy, our results still proved that mediators associated with RAS might influence the modulation of MMPs and TIMPs in DOX-induced acute cardiomyopathy and the modulation might be related to the pERK1/2 and pSTAT3 signaling pathway.