心房顫動組織與血管加壓素處理對心肌纖維母細胞中MMPs和TIMPs之表現調節

Abstract

心房顫動（atrial fibrillation; AF）為常見的心律不整疾病。在AF病程中，常見因細胞外基質（ECM）的重塑（remodeling）造成的心房組織結構的病變，此種因ECM代謝失常而堆積於細胞間隙的病灶，稱之為纖維化（fibrosis）。已知心臟纖維母細胞中的基質金屬蛋白酶（matrix metalloproteinases; MMPs）與其組織抑制因子（tissue inhibitor of metalloproteinases; TIMPs）的表現調節，是心臟纖維化過程中調節膠原蛋白（collagen）分解的重要因子。然而，在持續性（sustained）AF病程中，MMPs與TIMPs對於ECM代謝調節機轉至今仍未清楚。因此，本實驗主旨在探討於持續性AF組織與經angiotensin II（Ang II）和angiotensin-(1-7)（Ang-(1-7)）處理的心臟纖維母細胞（cardiac fibroblast）中，collagen type I、MMP-2、MMP-9、TIMP-1及TIMP-2的表現調節情形。 在本研究中所使用的動物與細胞分別為經節律器激搏誘發（pacing-induced）持續性AF的Yorkshire-Landrace豬隻與大鼠心臟纖維母細胞株H9c2。我們一共由14頭AF與12頭正常竇律（sinus rhythm; SR：即sham control）豬隻取得心房組織。實驗方法則分別以Masson’s trichrome染色與西方墨點法來檢測心房組織中ECM與collagen type I蛋白表現量；利用gelatin zymography分析MMP-2與MMP-9酵素活性；以半定量RT-PCR方法測定collagen type I、MMP-2、MMP-9、TIMP-1及TIMP-2的mRNA表現量。 實驗結果顯示，在AF心房的細胞間隙中有顯著組織纖維化病灶（AF, 21.6 ± 3.3%; SR, 5.2 ± 1.7%, p < 0.01）；在AF心房組織中的collagen type I（1.79 ± 0.14）也顯著高於SR組織中者（1.17 ± 0.26）(p < 0.01)。在AF心房組織中MMP-9的酵素活性與mRNA表現量分別較SR組織高出7.1倍（p < 0.01）及4.3倍（p < 0.01）；但是MMP-2活性，MMP-2、TIMP-1及TIMP-2的mRNA表現量在AF和SR組織中則相似。在心臟纖維母細胞的實驗結果顯示，經Ang II和Ang-(1-7)（10-9, 10-7及10-5 M）處理的H9c2細胞之MMP-2活性皆下降（p < 0.05）；另外在10-7 M的Ang-(1-7)處理下，可顯著誘發collagen type I 與MMP-9的mRNA表現量（p < 0.05）。 本研究中，我們發現在持續性AF組織中有高度纖維化現象，並伴隨著高量collagen type I與MMP-9的表現，但是並不影響MMP-2、TIMP-1及TIMP-2的基因表現。我們認為在AF發生組織結構性重塑病程中，可能與心臟纖維母細胞調控MMP-9表現的機制有關；而當MMPs與TIMPs的平衡關係被破壞時，其會影響ECM代謝的異常，可能造成持續性AF組織的纖維化病變。

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Structural abnormality is usually accompanied with extracellular matrix (ECM) remodeling process of AF. During structural remodeling of AF, abnormal ECM metabolism in the atrial tissues yields extensive interstitial matrix accumulation, a process termed fibrosis. Matrix metalloproteinases (MMPs) and their natural inhibitor of tissue inhibitor of metalloproteinases (TIMPs) are expressed in the cardiac fibroblasts have been shown to play important role in the regulation of collagen degradation in cardiac fibrosis. However, it is still unclear that mechanisms of MMPs and TIMPs in ECM metabolism during sustained AF. Therefore, the aims of this study was to explore altered collagen type I, MMP-2, MMP-9, TIMP-1 and TIMP-2 expression in the atrial tissues with sustained AF and in the cardiac fibroblasts treated with angiotensin II (Ang II) and angiotensin-(1-7) (Ang-(1-7)). Yorkshire-Landrace pigs with pacing-induced sustained AF and sinus rhythm (SR; i.e., sham control) and a cardiac fibroblast H9c2 cell line of rat were used in this study. Atrial tissues were obtained from 14 pigs with sustained AF and 12 pigs with SR. The ECM was investigated by Masson’s trichrome stain. Western blot was used to analyze the protein expression of collagen type I and gelatin zymography was used to assay the enzyme activity of MMP-2 and MMP-9. The gene expression of collagen type I, MMP-2, MMP-9, TIMP-1, and TIMP-2 were assayed by quantitative RT-PCR. The myocardial fibrosis markedly accumulated in the interstitial cardiomyocytes of atrial tissues with AF (AF, 21.6 ± 3.3%; SR, 5.2 ± 1.7%, p < 0.01). The content of collagen typeⅠin the AF subjects (1.79 ± 0.14) was also increased as compared with that in the SR subjects (1.17 ± 0.26) (p < 0.01). MMP-9 activity and expressed MMP-9 mRNA in the AF was higher than that in the SR by 7.1 folds (p < 0.01) and 4.3 folds (p < 0.01), respectively. However, MMP-2 activity and expressed MMP-2, TIMP-1 as well as TIMP-2 mRNA in the AF and SR subjects were insignificantly different. In the cardiac fibroblast H9c2 cells, the MMP-2 activity was decreasing for both Ang II and Ang-(1-7) (10-9, 10-7 and 10-5 M) treatments (p < 0.05). The mRNA levels of collagen type I and MMP-9 in the cells treated with 10-7 M Ang-(1-7) could be significantly induced (p < 0.05). In this study, we demonstrated the up-regulation of MMP-9 and collagen type I in the AF atria with fibrosis, but no significantly different on the mRNA levels of MMP-2, TIMP-1 and TIMP-2. We supposed that a role of structure remodeling in the AF is associated with MMP-9 regulated in cardiac fibroblasts. Moreover, imbalance of MMPs and TIMPs influences the ECM metabolism that may contribute in the process of sustained AF.