心房顫動症之分子重塑：心房基因表現及基質金屬蛋白酶與其抑制因子之調節

Abstract

心房顫動症（atrial fibrillation; AF）是臨床上最常見的心律不整疾病。AF主要的病理特徵為心房重組現象，其可被分為電性、收縮間期及結構性重組等。經由這心房組織之病變，AF能具有自我維持（self-sustaining）及持續性進展（progressive disease）的特性。然而，在AF病程中這些心房重組現象之分子機制並不明確，若能深入了解AF病程之分子機轉與其調控機制，必將對此心律不整疾病之控制及臨床的治療研究能有相當助益。因此，本研究以快速心博節律器誘導（rapid atrial pacing-induced）持續性AF的豬隻作為實驗模式，偵測AF豬隻之心房組織基因表現改變，同時檢測心房組織中基質金屬蛋白酶（matrix metalloproteinases; MMPs）和金屬蛋白酶組織抑制因子（tissue inhibitors of metalloproteinases; TIMPs）的表現與活性，以探討在AF病程中組織病變的分子機轉。 本論文分成兩部分，第一部分以低密度AF基因晶片測定AF之心房組織的基因表現差異。結果顯示AF心房組織中，31個差異性表現的基因主要有參與轉錄調控、訊息傳遞和細胞結構等重要基因。其中，four and a half LIM domains protein-1（FHL1）、transforming growth factor (TGF)-β-stimulated clone 22（TSC-22）及cardiac ankyrin repeat protein（CARP）等重要轉錄調控因子，其表現量上升最為顯著，另外chromosome 5 open reading frame gene 13（P311）於AF心房組織中表現量則大幅下降。FHL1和CARP在心肌疾病的病變機轉中，扮演轉錄調控及肌纖維（myofilament）組成因子之重要角色。我們也證實腎上腺素受體促進劑（□-adrenergic agonist）能調控心臟H9c2細胞株之FHL1和CARP基因表現。另一方面，AF心房TSC-22的高表現和P311顯著下降則顯示TGF-□訊息傳遞之增強，我們進一步檢示出TGF-□1及TGF-□2 mRNA於AF豬隻心房組織有高量表現，這表示了TGF-□在AF心房重組現象中扮演重要角色。據此，我們推測AF病程中，TGF-□及腎上腺素受體訊息傳遞路徑的活化參與了心房重組之分子機制。 第二部分則是針對AF病程中細胞外基質（extracellular matrix; ECM）重組機制之研究。此部分實驗結果主要為基質金屬蛋白酶（metalloproteinases; MMPs）與其抑制因子（tissue inhibitors of MMPs ;TIMPs）在AF心房組織中的表現位置與表現態樣（profile）之改變。我們發現gelatinases在AF心房組織中的活性表現顯著上升，推測其可能參與TGF-□1的活化，進而促使心房ECM重組及纖維化。再者，AF組織中TIMPs顯著上升，顯示出抑制pro-MMP9活化及抑制MMPs活性的能力提高。另一方面，我們發現AF心房中TIMP-1表現與gelatinases活性位置分佈相似，然而只有少部分TIMP-3與gelatinases活性存在同一區域，並顯示出TIMP-3抑制心肌細胞周圍gelatinases之活性。此結果表示TIMP-1和TIMP-3於AF心房組織之重組過程可能展現不同抑制功能。本研究中，AF心房組織MMPs和TIMPs表現情形與彼此間交互關係之實驗結果，將有助於了解ECM重組與心房顫動之病理機制。

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in clinical practice. AF appears to be a progressive disease and to be self-sustaining through alterations in atrial tissue properties. The processes leading to the worsening of AF over time were characterized by electrical, contractile and structural changes, referred to as atrial remodeling. However, the underlying mechanisms involved in the remodeling of atria with AF are incompletely defined. Additionally, the knowledge of molecular mechanisms responsible for the atrial remodeling of AF inevitably require further investigation to improve the clinical management of AF and the efficacy of therapy. In this study, a rapid atrial pacing (RAP)-induced AF model was employed to investigate the alteration of the gene expression profile and the expression and activity of matrix metalloproteinases or tissue inhibitors of metalloproteinases in the atria with AF for addressing the remodeling processes at molecular level. This thesis is comprised of two parts. The first one investigates gene expression responses obtained from a low-density cDNA array in the porcine atria with fibrillation. We identified 31 genes involved in transcriptional regulation, signal transduction or structural components, which were either significantly upregulated or downregulated in the atria with AF. The genes for four and a half LIM domains protein-1 (FHL1), transforming growth factor-β (TGF-β)-stimulated clone 22 (TSC-22), and cardiac ankyrin repeat protein (CARP) were significantly upregulated, and chromosome 5 open reading frame gene 13 (P311) was downregulated in the fibrillating atria. FHL1 and CARP play important regulatory roles in cardiac remodeling by transcriptional regulation and myofilament assembly. Induced mRNA expression of both FHL1 and CARP was also observed when cardiac H9c2 cells were treated with an adrenergic agonist. Increasing TSC-22 and marked P311 deficiency could enhance the activity of TGF-β signaling and the upregulated TGF-β1 and -β2 expressions were identified in the fibrillating atria. The results presented in the first part suggest that observed alterations of underlying molecular events were involved in the rapid-pacing induced AF, possibly via activation of the β-adrenergic and TGF-β signaling. The second part focuses on the mechanisms responsible for the atrial extracellular matrix (ECM) remodeling in atrial fibrillation. The major findings presented in this part indicated the localization and alteration in profile of matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) expression in the atrial myocardium with AF. The striking increase in gelatinase activity was found in the AF, which might be associated with the activation of TGF-□1 and contribute to ECM remodeling and fibrosis in the atrium. In addition, the increase in TIMP inhibitory activity in the fibrillating atria may provide regulation of proMMP-9 activation and inhibition of the activated MMPs through their inhibitory ability or complexes with proMMP-9. Another important finding was that TIMP-1 mostly colocalized with gelatinase activity over the AF tissues, showing the coexistence of gelatinase activity and TIMP-1; however, TIMP-3 appeared only partial colocalization and to discontinue the gelatinase activity surrounding the cardiomyocytes, revealing that TIMP-1 and TIMP -3 may play a differential role in inhibiting the gelatinase in vivo. The identification of changes in certain species of MMP and TIMP as well as their in vivo interplay in the RAP-induced AF model may improve understanding of the pathophysiology of atrial remodeling and fibrillation.