阿黴素誘發心肌病小鼠之心臟組織中基質金屬蛋白酶

Abstract

心臟結構與功能上之失調是心臟衰竭（heart failure, HF）臨床上的表徵，其為常見之病症，以及造成死亡的普遍原因。異常之細胞外基質（extracellular matrix, ECM）重塑（remodeling）被認為在HF病程之結構性異常中扮演著重要的角色。基質金屬蛋白酶（matrix metalloproteinases, MMPs）是一類活性依賴於Zn2+等金屬離子且參與降解ECM的蛋白酶家族，其與內源性的MMPs組織抑制因子（tissue inhibitor of metalloproteinases, TIMPs）精密調控著組織中ECM的代謝。許多證據顯示多種心血管疾病與MMPs和TIMPs間的表現失調相關。阿黴素(doxorubicin, Dox）廣泛使用於腫瘤之臨床治療，但其應用受到給予劑量累積可能導致的心臟毒性，以致引發心肌病而有所限制。據此，我們利用Dox誘發HF的小鼠動物模式來檢視此心肌病病程中MMPs及TIMPs的表現樣態。 在本研究中，C57BL/6J小鼠每週以腹腔注射4 mg/kg劑量的Dox直到20 mg/kg的累積劑量。小鼠的左心室將分別在第一劑給藥之後的第1, 3, 5, 9及12週取出，以用於MMP-1、MMP-2、MMP-9及TIMPs表現樣態之分析。根據心電圖與HF生物性指標分析，S-T 區間的增長與心臟組織利鈉激素A（atrial natriuretic peptide）及利鈉激素B（brain natriuretic peptide）的mRNA上升顯示出Dox心臟毒性的效應。於基因表現上，明膠酶（gelatinase）（包含MMP-2與MMP-9）基因的表達在此疾病模式後期有下降的趨勢，然而膠原蛋白（collagen）與TIMPs的基因表現呈現穩定狀況。在蛋白質分析中顯示，雖然整體MMP-1的蛋白質表現量並沒有受到Dox處理的影響，但活化態的MMP-1（act-MMP-1）隨著處理時間的延長有逐漸增加的現象。藉由膠內酶譜（in-gel zymography）分析，結果顯示MMP-9活性在第一週有上升的情形，其後隨著病程的延長，gelatinase活性呈現下降，此現象與基因表現上的樣態趨勢類似。然而，與gelatinase活性的變化相反，TIMPs的活性是隨著HF病程時間的延長而上升。 我們演示了Dox誘發之心肌病模式中小鼠左心室心肌內MMPs和TIMPs之時間相依性之變化。在本研究結果中可歸納出幾項值得注意的發現，此包含MMP-1活化的漸進增加、gelatinase 基因的表達下降、gelatinase活性降低及TIMPs活性的上升。這些心臟組織中的MMPs及TIMPs變化樣態可助於釐清Dox誘發之心肌病中心臟結構重塑的機轉。

Heart failure (HF), a common cause of morbidity and mortality, is a clinical syndrome characterized by structural and functional cardiac disorder. Abnormal metabolism of extracellular matrix (ECM) has been proposed to participate in structural remodeling during the progressive development of HF. ECM metabolism tissue is precisely modulated by matrix metalloproteinases (MMPs) and their native inhibitors, tissue inhibitor of metalloproteinases (TIMPs). MMPs constitute a family of extracellular zinc-dependent endopeptidases and can degrade essentially ECM components. Various evidences have shown that several cardiovascular diseases are associated with imbalance of MMPs and TIMPs. Doxorubicin (Dox) is widely used for clinical treatment of a variety of malignancy. Usefulness of Dox is limited, however, by its cardiotoxicity which may result in cardiomyopathy in a dose-dependent manner. Thus, we utilized Dox to induce HF and examined the expression profiles of MMPs and TIMPs during the process of chronic cardiomyopathy in a mouse model. In this study, C57BL/6J mice were intraperitoneal injection with 4 mg/kg of Dox weekly until reaching the accumulated dosage of 20 mg/kg. The left ventricles (LV) of animals were harvested at week 1, 3, 5, 9 and 12 after the first injection for examining the expression profiles of MMP-1, MMP-2, MMP-9 and TIMPs. According to the analyses of electrocardiogram and HF biomarker, increased S-T interval and up-expressed mRNA of atrial natriuretic peptide and brain natriuretic peptide indicated the effects of Dox cardiotoxicity. At transcriptional level, the transcripts of gelatinase (MMP-2 and MMP-9) were down-expressed but collagen and TIMPs expression were stable during the end stage of model. At protein expression level, total MMP-1 was not influenced by Dox treatment but active form of MMP-1 (act-MMP-1) was progressively increased over time. By in-gel zymography analysis, the increased MMP-9 activity was detected at first week and then gelatinase activity during end stage was reduced as similar as the gene expression profile. In contrast to gelatinase, the TIMPs activity was increased according to the measurement of reverse zymography. In conclusion, we demonstrated a time-dependent alteration of MMPs and TIMPs in mouse LV with Dox-induced cardiomyopathy. Several notable findings were discovered including gradual increase of MMP-1 activation, reduction of mRNA and enzyme activity of gelatinase, and induction of TIMPs activity in the Dox-induced cardiomyopathy. These expression profiles of the myocardial MMPs and TIMPs may provide insight into the mechanism of cardiac remodeling in Dox-induced cardiomyopathy.