心肌缺氧對心臟分子生物及電氣生理之角色探討：從基礎到臨床研究

The role of hypoxia in cardiac molecular biology and electrophysiology remodeling,from bench to clinical practice

蔡適吉(Su-Kiat Chua)

指導教授 ： 林裕峯

共同指導教授 ： 連立明(Li-Ming Lien)

臺北醫學大學/醫學院/臨床醫學研究所/博士(2016年)

摘要

缺氧與心臟分子生物學和電生理學的重塑相關。本研究的目的是探討心肌細胞對缺氧的反應及其潛在的調節分子機制。此外，心房顫動是缺血性心臟病中最常見的心律不整之一。因此，在本臨床研究中，我們主要的目的是探討接受心臟手術的缺血性心臟病患者發生術後心房顫動的風險和預測因素。
Chemerin是一種新型脂肪素，在血管內皮細胞的炎症狀態扮演重要的角色。缺氧會引起內皮細胞增殖，遷移和血管生成。因此，我們的基礎的研究旨在評估（1）人類冠狀動脈內皮細胞暴露於缺氧後，Chemerin蛋白質和mRNA於缺氧人類冠狀動脈內皮細胞中的表現。 Chemerin蛋白水平在2.5％O 2下缺氧4小時後明顯增加。同時腫瘤壞死因子α（TNF-α）亦在缺氧1小時後達到峰值。在缺氧期間和外源性添加的TNF-α都可以刺激chemerin的表現，而ERK抑制劑（PD98059），ERK siRNA或抗TNF-α抗體都會減弱由缺氧誘導chemerin的表現。EMSA測定發現缺氧會增加transcription factor SP1之間DNA-蛋白結合。Luciferase assay證實在缺氧期間， chemerin promoter中SP1的轉錄活性亦明顯的增加。缺氧顯著增加了人類冠狀動脈內皮細胞的遷移（和管腔形成），而PD98059，抗TNF-α抗體和chemerin siRNA各自減弱了這些作用。根據上述研究結果，缺氧激活人類冠狀動脈內皮細胞中的chemerin表達。缺氧誘導的chemerin由TNF-α介導並且至少部分由ERK途徑介導。而且缺氧透過Chemerin而促進人類冠狀動脈內皮細胞增加血管的生成。
缺血性心臟病病患在接受心臟手術後發生術後房顫（POAF）預示著較高的短期和長期死亡率。然而，目前沒有評分系統來預測術後房顫的發生。本研究的目的是探討（2）CHADS2和CHA2DS2-VASc評分是否可以成為缺血性心臟病患者經心臟手術後發生術後房顫有用的風險評估工具。腎功能不全，左心室舒張功能障礙與房顫的發生率相關。接下來的研究在探討（3）腎功能不全，左心室舒張功能障礙和術後房顫之間的關聯，（4）添加腎功能不全於評分系統可以提高CHA2DS2-VASc評分預測術後房顫的準確性。
在我們的第二項研究中，共有277例連續接受心臟手術的患者被前瞻性地納入這項風險分析研究。我們從收集的數據計算CHADS2和CHA2DS2-VASc分數，並預測心臟手術後30天內術後房顫的發生。在心臟手術後，有84例（30％）的患者發生術後房顫，中位數為2天（範圍，0-27天）。在單獨的多變量回歸分析中，CHADS2和CHA2DS2-VASc評分是術後房顫的顯著預測因子。CHADS2和CHA2DS2-VASc得分≥2的Kaplan-Meier分析比分數<2（both log rank, p < 0.001）有更高術後房顫的發生率。此外，CHA2DS2-VASc評分在截斷值為2，可進一步將CHADS2評分為0或1的患者分成2組，並有不同術後房顫的發生率（12％vs. 32％，p = 0.01）。
在我們的第三項研究中，265名接受心臟手術的缺血性心臟病病患被前瞻性地納入研究。在心臟手術之前進行心臟超音波。根據腎小球濾過率（GFR）分為3組（第1組，≥90mL·min -1·1.73 m-2; 第2組,60-90 mL·min -1·1.73 m-2;和第3組，<60mL·min-1.·1.73m-2）。 術後房顫發生在83例265例（31.3％）。新發術後房顫的發生率從第1組的15.2％（79/12）增加到第2組的27.8％（97/27），第3組的49.4％（44/89）（p <0.001）。此外，隨著腎功能障礙從1組增加到3組，LV舒張功能障礙的發生率定義為E / e'> 15（第1組：19.0％，第2組：38.1％，第3組：48.3％ p <0.001）。 GFR的絕對值與E / e'比值顯著相關（r = -0.39，p <0.001）。不同腎功能的組別在多變量分析中仍為術後房顫的獨立預測因子（[OR]，1.90; 95％信賴區間[CI]，1.26-2.87; p = 0.002）。
在第四個研究中，我們招募了350名接受心臟手術的缺血性心臟病患者。腎功能不全被定義為估計腎小球濾過率（eGFR）<60 mL·min -1·1.73 m-2。所有患者在住院期間通過連續心電圖遙測監測術後房顫的發生。350例患者中有103例發生POAF（29％）。與無術後房顫相比術後房顫患者有較長的重症監護病房住院天數（3.7±2.2天對比3.1±1.4天，P = 0.002）。在多變量分析中，CHA2DS2-VASc評分和腎功能不全都是術後房顫的獨立預測因子。腎功能不全可以進一步將CHA2DS2-VASC評分為0或1的病患分為具有不同POAF率的兩組（3.1％對68.8％，P <0.001）。一個新的評分系統（R- CHA2DS2-VASC評分）通過加入腎功能不全於CHA2DS2-VASC評分可提高其預測的準確性。AUC曲線下的面積從0.68增加到0.71（p <0.001）。此外，左心室舒張功能障礙的比率也隨著腎功能不全而增加。
綜合以上研究，CHADS2，CHA2DS2-VASc評分和腎功能不全可作為缺血性心臟病患者發生術後房顫的預測因子，而可能有助於在術識別高危患者。腎功能不全與左心室舒張功能障礙相關可能是術後房顫的重要危險因素。

關鍵字

趨化素 ； 缺氧 ； 心房顫動 ； 腎功能不全

並列摘要

Hypoxia is associated with cardiac molecular biology and electrophysiology remodeling. The aim of the current study is to investigate the responses of cardiomycytes to hypoxia and its underlying regulatory molecular mechanism. In addition, atrial arrhythmia, such as atrial fibrillation is one of most common comorbidity in ischemia heart disease. Therefore, in our clinical study, we aim to investigate the risk and predictors of atrial fibrillation in patients with ischemic heart disease receiving cardiac surgery.
Chemerin, a novel adipokine, plays a role in the inflammation status of vascular endothelial cells. Hypoxia causes endothelial-cell proliferation, migration, and angiogenesis. Our basic study was aimed at evaluating (1) the protein and mRNA expression of chemerin after exposure of human coronary artery endothelial cells (HCAECs) to hypoxia. Cultured HCAECs underwent hypoxia for different time points. Chemerin protein levels increased after 4 h of hypoxia at 2.5% O2, with a peak of expression of tumor necrosis factor α (TNF-α) at 1 h. Both hypoxia and exogenously added TNF-α during normoxia stimulated chemerin expression, whereas an ERK inhibitor (PD98059), ERK small interfering RNA (siRNA), or an anti-TNF-α antibody attenuated the chemerin upregulation induced by hypoxia. A gel shift assay indicated that hypoxia induced an increase in DNA-protein binding between the chemerin promoter and transcription factor SP1. A luciferase assay confirmed an increase in transcriptional activity of SP1 on the chemerin promoter during hypoxia. Hypoxia significantly increased the migration of (and tube formation by) HCAECs, whereas PD98059, the anti-TNF-α antibody, and chemerin siRNA each attenuated these effects. In summary, hypoxia activates chemerin expression in cultured HCAECs. Hypoxia-induced chemerin expression is mediated by TNF-α and at least in part by the ERK pathway. Chemerin increases early processes of angiogenesis by HCAECs after hypoxic treatment. In summary, hypoxia activates chemerin expression in cultured HCAECs. Hypoxia-induced chemerin expression is mediated by TNF and at least in part by the ERK pathway. Chemerin increases early processes of angiogenesis by HCAECs after hypoxic treatment
The presence of postoperative atrial fibrillation (POAF) predicts a higher short- and long-term mortality rates. However, no scoring system was used to identify patients at high risk of POAF. The aim of our study was to investigate (2) the CHADS2 and CHA2DS2-VASc scores were useful risk assessment tools for new-onset POAF after cardiac surgery. Renal dysfunction is associated with a higher rate of atrial fibrillation in the clinical practice. The aim of our following study aimed to investigate (3) the associations among renal function, left ventricular (LV) diastolic dysfunction, and postoperative atrial fibrillation (POAF), (4) the addition of renal dysfunction into the scoring system could improve diagnostic accuracy of the CHA2DS2-VASc score to predict POAF.
In our second study, a total of 277 consecutive patients who underwent cardiac surgery were prospectively included in this risk stratification study. We calculated the CHADS2 and CHA2DS2-VASc scores from the data collected. The primary end point was the development of POAF within 30 days after cardiac surgery. Eighty-four (30%) of the patients had POAF at a median of 2 days (range, 0–27 days) after cardiac surgery. The CHADS2 and CHA2DS2-VASc scores were significant predictors of POAF in separate multivariate regression analysis. The Kaplan-Meier analysis based on the CHADS2 and CHA2DS2-VASc scores ≥ 2 obtained a higher POAF rate than when based on scores < 2 (both log rank, p < 0.001). In addition, the CHA2DS2-VASc scores could be used to further stratify the patients with CHADS2 scores of 0 or 1 into 2 groups with different POAF rates at a cutoff value of 2 (12% vs. 32%, p = 0.01).
In our third study, 265 consecutive patients who underwent cardiac surgery were prospectively enrolled in the study. Echocardiography was performed before cardiac surgery. The patients were divided into 3 groups based on their glomerular filtration rate (GFR) (group 1,≥90 mL·min-1·1.73m-2;group 2,60–90 mL·min-1·1.73m-2;and group 3,<60 mL·min-1·1.73m-2). POAF occurred in 83 of 265 patients (31.3%). The rate of new-onset POAF increased from 15.2%(12 of 79) in group 1 to 27.8%(27 of 97) in group 2 and 49.4%(44 of 89) in group 3(p<0.001). Further, with increasing renal dysfunction from groups 1 to 3, the rate of LV diastolic dysfunction—defined as E/e′ > 15—also increased (group 1: 19.0%, group 2: 38.1%, and group 3: 48.3%; p<0.001). The absolute value of GFR was significantly correlated with E/e’ ratio (r = −0.39, p<0.001). The groups of different renal function remained as the independent predictor of POAF in the multivariate analysis (odds ratio [OR], 1.90; 95% confidence interval [CI], 1.26–2.87; p=0.002).
In forth study, we enrolled 350 consecutive patients who underwent cardiac surgery. Echocardiography was performed before cardiac surgery. Renal dysfunction was defined as estimated glomerular filtration rate (eGFR) < 60 mL·min-1·1.73m-2. All patients were monitored with continuous electrocardiographic telemetry for the occurrence of POAF until the day of hospital dismissal. POAF occurred in 103 of 350 patients (29%). Patients with POAF was associated with longer intensive care unit stay compared with those without POAF (3.7 ± 2.2 days vs. 3.1 ± 1.4 days, p = 0.002). Both the CHA2DS2-VASc score and renal dysfunction were independent predictors of POAF in multivariate analysis. Renal dysfunction can further stratify patients with a CHA2DS2-VASc score of 0 or 1 into two groups with different POAF rates (3.1% vs. 68.8%, p < 0.001). A new scoring system (R- CHA2DS2-VASc score) derived by assigning an additional point representing renal dysfunction to the CHA2DS2-VASc score could improve its predictive accuracy. The area under the receiver operating characteristic curve increased from 0.68 to 0.71 (p < 0.001). Furthermore, the rate of left ventricular diastolic dysfunction also increased with increasing renal dysfunction.
In summary, CHADS2, CHA2DS2-VASc scores and decreased GFR were predictive of POAF after cardiac surgery and may be helpful for identifying high-risk patients. Renal dysfunction, associated with left ventricular diastolic dysfunction, was a significant risk factor for POAF after cardiac surgery and may improve the diagnostic accuracy of the CHA2DS2-VASc score.

並列關鍵字

Chemerin ； Hypoxia ； Atrial fibrillation ； Renal dysfunction

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