臺灣博碩士論文加值系統

背景和目的：慢性、過度酒精的使用及所產生的毒性代謝產物，逐漸被認為會對心臟的結構和功能造成一定程度的危害。因此，在心臟呈現明顯心肌病變及射出分率異常前在臨床上能找出臨床前期心臟功能危害的證據，因而改變個體飲酒行為並更進一步預防續發心衰竭或心房顫動(AF)的風險有其重要性，特別是在那些長期習慣性輕到中度劑量酒精使用的個體。
研究方法：我們利用北區一家醫學中心(台北馬偕醫院)持續進行的心血管健康篩檢診查資料庫中，從中篩選出從2009 年 6 月至2011 年12 月完整接受心臟超音波功能評估、基礎人體測量學資料及相關生理檢查的成年個體，總共有3,946 位(均年齡：49.9 歲，35%女性)。其中心臟超音波評估包括高解析度組織學(Tissue Doppler)心臟超音波，及左心室型變(Strain [S])和心房型變及型變率(Strain rate [SR])等。參與的研究個體並根據飲酒行為及習慣分為三個族群：1) A 組，無使用酒精組：完全不使用或每周少於1 次(個體數：3,464)；2) B 組，輕度酒精使用組：每周1 到6 次(個體數：323)；3) C 組，中度酒精使用組：每周6 次或以上(個體數：89)，並排除懷疑濫用者2 位；以及4) D 組，曾經使用病已經戒酒者(個體數：70)。我們採取ANOVA 組間多重比較測定、及多變項飲酒行為劑量反應關係(dose-response relationship)和心肌結構及功能變化做相關性評估。我們並進一步建立酒精性心臟功能缺損之小鼠動物模式，以雄性四個月大之C57BL/6 小鼠，依照酒精飼料的餵食與否分為三組，包括：1)正常對照飼料[Ctrl]; 2)4% 或3)6%液態酒精飼料組作為唯一食物及水源攝取來源持續餵食十四周。以光學電波定位系統(optical mapping)來量化心肌之電傳導速度，並以免疫組織螢光染色法(Immunohistochemistry)、蛋白免疫抗體學(Immunoblotting)並輔以免疫共軛焦顯微鏡(Confocal)來進一步探討心肌的發炎及纖維化程度(fibrosis)、聯結蛋白43(Cx43)及電壓依賴型鈉離子(Nav1.5)通道表現量、分布以及型態改變在酒精餵食小鼠心臟功能的影響。

主要發現:1. 較大的左心室內徑、較厚的心室壁及較大的心室質量、較長的左心室返流減速時間(DT)、等容舒張時間间(IVRT)、及較低的左心室舒張速度(E’)伴隨著飲酒習慣的週頻率增加而上升 (ANOVA p<0.001)，但是左心室射出分率相對維持不變(p: 0.345)。
另外，較低的左心室收縮速度(S’)在飲酒組也顯著下降 (p: 0.0092)；
2. 下降的左心室縱向收縮功能，以平均縱向型變(GLS)來評估，也明顯顯示出組間差異(A 組 vs B 組/C 組： -18.32±2.29% vs -18.11±2.3%/-17.94±2.39%) (ANOVA p<0.001)；同樣的，左心房型變(LA S)及早期舒張期型變率(LA SRe)也呈現顯著分級性的下降。整體而言，比起無使用酒精組，輕度和中度酒精使用的個體在多變項分析分別呈現顯著下降的平均縱向型變、較低的左心房收縮型變率和明顯變差的左心房早期舒張期型變率(all p<0.001)。以傾向分數配對(Propensity Matching)檢定的方式進行1,140 位配對的參與者(2:1 分派，均年齡: 50.1±10.3 歲；無使用酒精組：760 位；輕度及中度酒精使用組：380 位)，比起無使用酒精組(760 位)，輕度及中度酒精使用組(380 位)同樣呈現明顯的功能改變；
3. 動物的結果顯示慢性餵食液態酒精飼料組的小鼠呈現增加的心室組織纖維化、第一型膠原蛋白( Collagen Type I )堆積、明顯的巨噬細胞浸潤及收縮功能下降，尤其在6%酒精飼料組。光學電波定位系統(optical mapping)顯示了漸進式的心室傳導擾亂(conduction disturbance)，包括較慢的傳導速度及異質性傳導的型態。蛋白免疫抗體檢測顯示了酒精飼料餵食的小鼠群同時呈現了減少(近50%)並且側移的聯結蛋白43(Cx43)表現，合併下調且功能異常之群聚型的鈉離子(Nav1.5)通道變化。
結論：本研究的結果發現習慣性飲用酒精的個體，即便是在每週使用頻率較輕或中度的劑量，也可以用較敏感的心肌型變方法偵測到左心室及左心房的功能異常；此外，功能失常的程度和酒精使用頻率遵循著劑量反應相關性並且獨立於其生活型態和臨床基礎特質。動物實驗藉由分子生物學的方式進一步解釋了部分心臟心肌收縮功能失常及潛在電生理變化的機轉在慢性酒精使用的小鼠模式。

Background and Objectives: Chronic excessive alcohol consumption may cause detrimental effects on cardiac structure and function. Evidence of preclinical LV and
left atrial (LA) functional alterations in alcohol consumers prior to the development of overt heart failure or AF may be clinically important based on the view point of primary preventive medicine, especially for light to moderate users.
Method: We consecutively studied 3,946 asymptomatic participants (mean age: 49.9 years, 35% female) from an ongoing cardiovascular health screening program at a
tertiary medical center in Northern Taipei, Taiwan. Tissue Doppler Imaging (TDI), left ventricular (LV) strain (S) and left atrial (LA) strain (S), strain rate (SR) were all
measured by echocardiography. Study participants were categorized based on graded alcohol consumption on a weekly basis: 1) Group A Non-drinkers: non-users or <1 drink/week, n=3,464; 2) Group B Light drinkers: 1-6 drinks/week, n=323; 3) Group C Moderate drinkers: >6 drinks/week, n=89; and 4) Group D Past drinkers (n=70). ANOVA with post hoc paired comparisons and multivariate regression models between dosage of alcohol consumption and LA/LV structural/mechanical changes were examined. Experimental animal model was conducted by randomizing male 4-month-old C57BL/6 mice into one of three groups: 1) liquid diet (control group [Ctrl]; or either a 2) 4% or 3) 6% alcohol liquid diet as their only fluid and food source for
continuous 14 weeks. Immunoconfocal imaging and Western immunoblotting analysis were performed to examine the myocardial tissue changes, with echocardiography and optical mapping used to assess altered cardiac structure/function and cardiac electrical conduction activation sequence and pattern.
Main Findings:
1. Larger LV dimension, wall thickness, increasing LA volumes, greater LV mass index, as well as more prolonged deceleration time (DT), isovolumic relaxation
time (IVRT) and lower’ relaxation E’ with relatively unchanged LV ejection fraction (LVEF) (p: 0.345) were observed across 3 alcohol consumption groups (ANOVA
p<0.001). Significant reduction of TDI-based myocardial contraction velocity S’was also observed (p: 0.0092);
2. A markedly worsened longitudinal systolic function in terms of lower averaged LV longitudinal strain (GLS) was observed (-18.32±2.29% vs -18.11±2.3%/-17.94±2.39%) in Non- vs Light/ Moderate drinkers (ANOVA p<0.001) (Coef: 0.25
[95% CI: 0.05 to 0.45] and 0.55 [95% CI: 0.17 to 0.92] with Non-Drinkers as reference group in multivariate models), which were accompanied by significant, graded reduction of LA strain (S) and LA diastolic strain rate (SRe) across 3
alcohol categories (all p<0.05). In general, worse GLS, LAS, SRe and SRs were observed in light-moderate drinkers compared to non-drinkers (all p<0.001), which followed a dose-response relationship. Finally, these differences did not change after careful 2:1 propensity-matching (n=760 for non- v.s. n=380 for light-moderate drinkers [n=307 and 73 for Light and Moderate Drinkers, respectively]);
3. In our experimental animal model, chronic alcohol-fed C57BL/6 mice, especially 6% mice, demonstrated substantially increased ventricular collagen I depots and fibrotic turnover, elicited macrophage infiltration of myocardium and contractile dysfunction. A specific perturbed and disturbed pattern and lower ventricular propagation velocity in both 4 and 6% alcohol mice groups were observed by optimal mapping (p<0.05). Down-regulated (nearly 50%), remodeled Cx43 together with clustered and diminished ordered membrane Nav1.5 distribution were also observed in mice feeding with alcohol diet, which in conjunction with increased cardiac fibrosis may explain the observed myocardial electromechanical dysfunctions.
Conclusions: Our findings suggest that habitual alcohol consumption, even at relatively low weekly doses, may be associated with detrimental effects on atrial and ventricular functions in a dose-response fashion independent of other clinical factors or life styles. Our animal model supported mechanistic insights into the pathological understanding of these electromechanical functional alterations at a cellular and molecular level.

致謝（Acknowledgement）....i
目錄....iii
English Abstract....v
Chinese Abstract....viii
List of Abbreviations....xi
Chapter 1 Introduction....1
1.1 Background....1
1.2 What are the possible mechanisms involved in alcohol-related
myocardial damage?....2
1.3 What is the potential method to assess subclinical myocardial function beyond chamber level alterations?....5
1.4 Would mild-to-moderate dose of alcohol use associated with certain left atrial/ventricular electromechanical disturbances?....6
1.5 What are the potential confounders in considering the biological effects on cardiac functional assessment for chronic ethanol users?....7
Chapter 2 Materials and Methods....8
2.1 Human Part....8
2.1.1 Database....8
2.1.2 Study cohort....9
2.1.3 Assessment of Conventional Echocardiography....9
2.1.4 Speckle-Tracking Analysis Protocol....10
2.1.5 Quantification and Categorization of Alcohol Consumption....13
2.1.6 Statistical Analysis....13
2.2 Animal Part....15
2.2.1 Preparation of Mice....15
2.2.2 Echocardiography Study for Mice....15
2.2.3 Optical Mapping Protocol....15
2.2.4 Immunoconfocal Microscopy and Image Analysis....16
2.2.5 Western Blotting....17
2.2.6 Statistical Analysis....18
Chapter 3 Results....19
3.1 Human Part....19
3.1.1 Baseline characteristics of study participants....19
3.1.2 Conventional Echocardiography Findings in Relation to Alcohol use....19
3.1.3 LV Deformation Alterations in Relation to Alcohol use....19
3.1.4 Results of Propensity Matching Analysis....21
3.2 Animal Part....21
3.2.1 Phenotypes of Alcohol Mice Model....22
3.2.2 Echocardiography and Electrocardiography Study Results....22
3.2.3 Results of Western Blotting and Immunoconfocal Findings....22
3.2.4 Results of Optical Mappings on Cardiac Electrical Conduction Velocity....23
Chapter 4 Discussion....24
4.1 Cardiovascular Effects with Light-moderate Levels of Alcohol
Consumption....24
4.2 The Association between Habitual Alcohol Consumption and LV Electromechanical Dysfunctions....25
4.3 The Association between Habitual Alcohol Consumption and Dynamic LA Mechanical Function....28
4.4 Summary....29
4.5 Study Limitations....29
References....33
Figures and Tables....43
Publications....68

1. Chung-Lieh Hung, Alexandra Gonçalves, Yu-Jun Lai, Yau-Huei Lai, Kuo-Tzu Sung, Chi-In Lo, Chuan-Chuan Liu, Jen-Yuan Kuo, Charles Jia-Yin Hou, Tze-Fan Chao, Bernard E. Bulwer Shing-Jong Lin, Hung-I Yeh, Carolyn S.P. Lam.
Light-moderate Habitual Alcohol Consumption is associated with Subclinical Ventricular and Left Atrial Mechanical Dysfunction in Asymptomatic Population: Dose-Response and Propensity Analysis. J Am Soc Echocardiogr. 2016;29:1043-1051. (IF = 6.852; Ranking = 13/126, CARDIAC & CARDIOVASCULAR SYSTEM) 第一作者
2. Chung-Lieh Hung, Shun-Chuan Chang, Sheng-Hsiung Chang, Po-Ching Chi, Yu-Jun Lai, Shih-Wei Wang, Yih-Jer Wu, Hung-I Yeh, Shing-Jong Lin, Che-Hong Chen, Daria Mochly-Rosen, Li-Yu Wang for the MAGNET Study Investigator. Genetic Polymorphisms of Alcohol Metabolizing Enzymes and Alcohol
Consumption are Associated with Asymptomatic Cardiac Remodeling and Subclinical Systolic Dysfunction in large Community-Dwelling Asians. Alcohol and Alcoholism. 2017 (Accepted in press) (IF = 2.76; Ranking = 9/34, SSI:
Substance Abuse) 第一作者
3. Chung-Lieh Hung, Chun-Ho Yun, Yau-Huei Lai, Kuo-Tzu Sung , Hiram G. Bezerra, Jen-Yuan Kuo, Charles Jia-Yin Hou, Tze-Fan Chao, Bernard E. Bulwer, Hung-I Yeh, Shou-Chuan Shih , Shing-Jong Lin , Ricardo C. Cury. An Observational Study of the Association among Inter-atrial Adiposity by Computed Tomography Measure, Insulin Resistance, and Left Atrial Electromechanical Disturbances in Heart Failure. Medicine. 2016;95:p e3912. (IF = 1.803) 第一作者