研究aliskiren和miR-26a對心肌梗塞引發傷害之保護效益及其相關機轉

Abstract

心肌梗塞(myocardial infarction)已經成為工業化與發展中國家的重要的健康問題，也是主要致死原因之一。雖然細胞凋亡已經被證實在心肌梗塞中扮演不可或缺的角色，而且也是心臟再塑化主要的原因之一，但是其詳細的機制以及有效的治療方法仍然尚未被釐清。Aliskiren是一種腎素的抑制劑（direct renin inhibitor, DRI），一種新開發的降血壓藥，近年來許多研究指出aliskiren可以減緩心肌梗塞後心臟傷害的情形，但詳細機制仍尚未明瞭。同時miR-26a在心肌梗塞中扮演的角色仍然存在許多爭議。本文主要目的在探討經過OGD (oxygen glucose deprivation)處理的H9c2心肌細胞以及在心肌梗塞模式的小鼠，aliskiren及miR-26a對心肌缺氧後對心臟保護的影響。 本論文分成兩個部分，第一部分，我們使用8到12週大的C57BL/6小鼠，藉由結紮心臟的左前降支冠狀動脈(left anterior descending coronary artery, LAD)做為心肌梗塞的動物模式。以皮下注射的方式給予aliskiren，並使用心臟超音波進行掃瞄，測量縮短分率，以評估小鼠的心臟功能。同時採用西方墨點法及免疫組織染色法，觀察aliskiren對心肌梗塞後心臟傷害相關蛋白表現的影響。在細胞模式中，我們使用OGD處理後的H9c2細胞給予aliskiren後，探討該治療是否有保護心肌細胞的能力以及其相關機轉。第一部分的結果顯示，在心肌梗塞動物模式中，aliskiren治療組降低cleaved caspase-3蛋白量、並提高LC3B-II的能力，同時藉由電子顯微鏡的檢測，證明aliskiren有增加自噬作用的能力。此外，我們利用H9c2細胞的缺氧缺糖模式，證實aliskiren不但能減少細胞凋亡、增加粒線體膜電位，也可以藉由增加自噬小體(autophagosome)的形成來增加自噬作用。我們也證實aliskiren增加心肌細胞的存活率是透過增加AMPK蛋白量的表現而增加自噬作用。 第二部分，我們同樣使用小鼠心肌梗塞的動物模式，以尾靜脈或心臟注射的方式給予miR-26a，並使用心臟超音波進行掃瞄測量fractional shortening (FS %)，以評估小鼠的心臟功能。同時採用西方墨點法及免疫組織染色法，觀察miR-26a對心肌梗塞後心臟傷害的保護效果以及與細胞凋亡相關蛋白的表現。同時我們想探討miR-26a所調控的標靶基因是否參與其中，我們進一步利用生物資訊的分析，找出其標靶基因為ataxia–telangiectasia mutated (ATM)，並在OGD處理後的H9c2細胞的模式中，給予miR-26a後，探討該治療是否有保護心肌細胞的能力以及與ATM蛋白的相關機轉。第二部分的結果顯示，我們利用螢光素酶檢測法(Luciferase Assay)證實miR-26a確實有調控其標靶基因ATM的能力，在OGD處理的心肌細胞中也能減少細胞凋亡以及其相關蛋白的表現量。我們也證實在心肌梗塞一天的小鼠中，miR-26a可以有效的減少ATM的表現以及減緩心肌細胞凋亡的狀況，而且在手術十四天後的老鼠中亦能改善其心臟功能，減少心臟纖維化的狀況以及collagen type I和connective tissue growth factor (CTGF)的表現量。此結果顯示aliskiren及miR-26a在細胞及動物實驗中皆可降低caspase-3的表現以及心臟纖維化的狀況。aliskiren改善缺氧缺糖引起心肌細胞的傷害是透過AMPK來活化自噬作用所致。而miR-26a則是透過調控標靶基因-ATM來控制。因此，aliskiren及miR-26a提供了一種治療心肌梗塞藥物的新選擇。

Myocardial infarction (MI) is a major health problem and the leading cause of death and disability in both industrialized and developing nations. Although cell apoptosis has been proposed to play the important role in the progression of MI and cardiac remodeling, its exact triggering mechanism is still unclear and the efficiency of the therapeutic approach is not available. Aliskiren, a direct renin inhibitor (DRI), was developed as an antihypertensive drug. The previous studies have indicated that aliskiren treatment attenuated cardiac remodeling after MI. However, the mechanism remained unclear. In addition, the role of miR-26a in MI is diverse and still controversial. The purpose of this study was to investigate the protective effects of aliskiren and miR-26a on oxygen glucose deprivation (OGD)-treated H9c2 cardiomyocytes and mouse model of MI. The doctoral dissertation is divided into two parts. In the first part, we used male C57BL/6 mice at age of 8-12 weeks and the left anterior descending coronary artery (LAD) was ligated as the MI animal model. In addition, the mice were injected subcutaneously with or without aliskiren. The cardiac function was evaluated by echocardiography. The percentages of fractional shortening (FS%) were measured. We investigated the effects of aliskiren on MI-induced injury and the related proteins were examined by western blot and immunohistochemical staining. Futhermore, we explored whether the aliskiren treatment protected cardiomyocytes and its related mechanisms in OGD-treated H9c2 cells. Aliskiren treatment reduced the protein expression of caspase-3 in MI mice. Moreover, aliskiren increased autophagy as demonstrated by LC3B-II expression and transmission electron microscopy. In OGD-treated H9c2 cells, aliskiren decreased apoptosis and improved mitochondrial membrane potential as well as increased autophagy via increased autophagosome formation. We also found that aliskiren induced cardiomyocyte survival through AMP-activated protein kinase (AMPK)-dependent autophagy. In the second part, we evaluated whether miR-26a ameliorated MI-induced injury. The miR-26a was injected into the myocardium or the tail vein after MI. The cardiac function was evaluated by echocardiography. The percentages of FS% were measured. We investigated that the effects of miR-26a on MI-induced injury and the related protein were examined by western blot and immunohistochemical staining. In addition, we examined whether the target gene of miR-26a participate in the pathway about MI-induced injury. Further analysis identified that ataxia–telangiectasia mutated (ATM) was a target gene for miR-26a based on a bioinformatics analysis. In addition, we explored whether the miR-26a treatment protected cardiomyocytes from OGD-induced injury and its mechanisms related to ATM. The luciferase assay showed that miR-26a overexpression significantly decreased the luciferase activity of the ATM 3′-UTR. MiR-26a significantly reduced ATM expression, apoptosis and apoptosis-related protein in OGD-treated H9c2 cells. MiR-26a effectively reduced ATM expression and cardiac apoptosis at day 1 after MI. Furthermore, we demonstrated that overexpression of miR-26a improved cardiac function and reduced cardiac fibrosis, which decreased both collagen type I and connective tissue growth factor (CTGF) expression in mice at day 14 after MI. Taken together, these results suggest that aliskiren and miR-26a reduced caspase-3 expression and improved cardiac fibrosis under in vitro and in vivo conditions. Aliskiren improved ischemia- and OGD-induced cardiac injury through activation AMPK-depentent autophagy. Moreover, miR-26a alleviated the development of MI by regulating ATM. Therefore, aliskiren and miR-26a may represent novel therapeutic agents in ischemic cardiac diseases.