李源德陳鵬生蘇銘嘉臺灣大學：劉言彬Liu, Yen-BinYen-BinLiu2007-11-272018-07-062007-11-272018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/55532背景 高膽固醇血症(hypercholesterolemia)在現代社會裡已被視為會對社會大眾健康造成顯著的威脅，也是心臟血管疾病的一個首要危險因子。降血脂治療在過去的研究中顯示可以有效地減少冠狀動脈疾病相關事件(coronary events)和病人的死亡率(all-cause mortality)。降血脂療的降低死亡率之療效有一部分可能是源自於對致命性心室心律不整(心室頻脈/心室顫動; ventricular tachycardia /fibrillation) 或心臟猝死的減少。Mitchell等學者企圖利用AIVD臨床試驗的病人來評估降血脂療法之抗心律不整療效，特別是其對心室頻脈/心室顫動發生率的影響。其中 AIVD(Antiarrhythmics Versus Implantable Defibrillators)臨床試驗是一個有關心室去顫器(Implantable cardiovertor defibrillator; ICD)的次級預防研究。其結果顯示在有動脈粥狀硬化的心臟疾病患者中，使用降血脂療法可以減少40% 心室頻脈/心室顫動發生的危險性。然而，我們仍不清楚降血脂療法其抗心律不整療效的詳細機轉為何? 心臟組織的異質性(tissue heterogeneity)在傳統的電生理學上被認為是造成電氣訊號或電波(electrical wavefronts)形成wavebreak及發生心室顫動的主要因素。組織的異質性常會因心臟疾病所造成的電生理或結構上的再塑(eletrical and structural remodeling)而大幅增加，進而造成電生理上的不穩定，引發心律不整。近年來的研究則指出一些動態的因素也會造成電生理上的不穩定，其中主要的有細胞的電生理特性及鈣離子調控二者。在電生理的特性方面，動作電位時間 (action potential duration, APD)及傳送速度(conduction velocity, CV)的restitution 特性、短期的心臟電生理記憶特性(cardiac memory)、電漫射電流(electrotonic currents)均會造成動態的電生理上的不穩定，而鈣離子調控則是另一個獨立的動態的因素。這些動態的因素可以和組織的異質性交互作用，進而使電氣訊號或電波形成wavebreak及發生心室顫動。除了電生理或結構上的再塑之外，陳鵬生教授的實驗用於近年來有一系列的研究說明了心臟新生芽神經(cardiac nerve sprouting)和心律不整之間的關係。根據陳教授的研究，神經再塑(nerual remodeling)在心律不整的發生上也是必須考慮之因素，其在心律不整的致病機轉上之重要性絕不亞於電生理或結構上的再塑。 大多數的心臟猝死病患均肇因於心室頻脈/心室顫動，而心肌缺血則是誘發這些致命性心室心律不整最常見的原因。急性心肌梗塞所造成的原發性心室顫動常伴隨著高死亡率，在這個情況下，想要有一個成功的心肺復甦術極為困難，也因此病人往往無法有良好的存活率。但是什麼樣的原理使一般的心肺復甦術於急性心肌缺血發生原發性心室顫動時，無法有效地救治病患則仍不十分清楚。進一步瞭解急性心肌缺血時心室顫動發生的致病機轉，使我們更有效地預防或治療心臟猝死應是十分重要的一步。在過去的研究中已指出心肌缺血會造成局部電生理特性的改變，包括細胞膜電位的去極化、細胞膜的興奮性(excitability)的減少、動作電位時間的縮短、傳送速度的減緩、及電生理不反應期(refractoriness beyond repolarization)的延長。我們知道動作電位時間的restitution特性及細胞的興奮性均對心室顫動有十分重要之影響，因此在過去的研究中將心室顫動分為兩種類型：第一型的心室顫動發生於有陡峭的動作電位時間restitution特性及正常的興奮性的心臟組織，呈現多發性小電波(multiple wavelets)形態，第二型的心室顫動則發生於有平緩的動作電位時間restitution特性及興奮性降低的心臟組織，呈現多空間時序上週期性(spatiotemporal periodicity)形態。當急性局部心肌缺血發生時，由冠狀動脈閉塞所產生的缺血區域，其動作電位時間restitution特性平緩且興奮性降低，而無缺血的區域，其興奮性正常且動作電位時間restitution特性則相對陡峭；因此，兩種不同類型的心室顫動很可能同時發生於急性局部心肌缺血時，進而增加了心肺復甦術困難度，以至於影響到病人的存活率。局部心肌缺血的動物模式，可以同時觀察缺血區域及無缺血的區域的電生理變化，以及兩區之間的交互作用，如此應可幫助我們進一步的了解心臟猝死的致病機轉。 本研究廣義及長期的研究目的是要測試下列假設：血脂異常會增加致命性心室心律不整發生的危險性，同時血脂異常也是構成心律不整造成心臟猝死的致病機轉上一個重要的基礎。在傳統的觀念中，血脂異常、高膽固醇血症會造成冠狀動脈的動脈粥狀硬化、血管內膜細胞功能異常、降低動脈粥狀班塊的穩定性(plaque instability)，進而引起心肌缺血及/或心肌梗塞。急性心肌缺血及/或心肌梗塞不僅是十分重要的心律不整誘發因子，同時也使心臟組織的異質性大幅增加，使心律不整更容易發生。我們將進一步探討急性局部心肌缺血時，缺血區域與無缺血的區域兩區之間的交互作用在心室顫動的致病機轉上之角色。除此之外，我們也認為高膽固醇血症也會直接影響心肌細胞的功能，因此進一步提出高膽固醇血症會引發心臟的神經及電生理再塑之假說，二者的交互作用是構成心律不整致病機轉上一個重要的基礎。也就是說，冠狀動脈及心臟組織均是血清膽固醇之標的作用器官(target organ)，高膽固醇血症所引起之心臟的缺血性變化、神經再塑、及電生理再塑均在心律不整發生的致病機轉扮演一定之角色。同時，我們也會對於急性冠心症的病患，評估血脂異常和致命性心室心律不整的臨床重要性。本研究將分為下列三個研究目標： 研究目標 1：探討於臨床心肌梗塞的病患中，血脂異常是否會增加致命性心室心律不整發生的危險性? 研究目標 2：利用高膽固醇血症的動物模式，研究高膽固醇血症是否會引發導致心律不整發生的心臟的神經及電生理再塑，進而增加致命性心室顫動發生的危險性? 研究目標 3：利用局部心肌缺血的動物模式，研究於急性局部心肌缺血時，第一型及第二型的心室顫動是否可能同時發生，並經由兩者之間的交互作用，增加心室顫動發生的機會及心室顫動在電生理上的複雜性? 材料與方法： 於研究目標1中，我們以58位於心肌梗塞第一天發生心室頻脈/心室顫動的病患為研究對象，並以另外58位沒有發生心室心律不整的心肌梗塞病患為對照組。兩組間的性別(104位男性)、年齡(平均年齡58 ± 10歲)、及血栓溶解療法(82例)的使用於收案時需對稱，所有研究對像之血中的脂質包括總膽固醇、高密度脂蛋白膽固醇(HDL-C)、低密度脂蛋白膽固醇(LDL-C)及三酸甘油脂均於心肌梗塞發生後的一週內及三個月後加以測量。其他的心血管疾病危險因子，臨床、血行動力學、及冠狀動脈血管攝影的特性也是本研究的評估項目。 於研究目標2中，我們以高膽固醇飼料或正常飼料分別餵食高膽固醇組(N=10, 6, 15, 25) 及對照組(N=10, 6, 15, 20)之紐西蘭白兔為期十二週(Protocol 1, 4-6)。在 Protocol 2中以高膽固醇飼料或正常飼料分別餵食高膽固醇組(N=12)及對照組(N=10)為期八週。在Protocol 3中則以不同的餵食方式觀察10隻紐西蘭白兔在不同血中高膽固醇濃度下的變化。我們對Protocol 1中的白兔於十二週的餵食期間內每兩週記錄一次十二導極心電圖，用於QTc和QTc dispersion的分析。對於Protocol 1和Protocol 2中的白兔，我們在餵食期間結束後會進行電生理檢查及螢光電位定位攝影(optical mapping)檢查，針對動作電位時間及其dispersion、傳導速度、及心室顫動發生之機率在高膽固醇飼料餵食後的變化加以研究。我們利用growth associated protein 43(GAP 43，神經新生的標記)和tyrosine hydroxylase(TH，交感神經的標記)的免疫細胞化學染色法以研究心臟的交感神經活性(Protocol 1和3)。 同時也利用123I-meta- iodobenzylguanidine(123I-MIBG) 攝影，對心臟的交感神經活性進行活體(in vivo)研究(Protocol 4)。而在Protocol 5中，我們則對心肌細胞之鈣離子流(calcium inward currents)其一些和鈣離子調控有關的蛋白表現[protein expression of Na+/Ca2+ exchanger (NCX) L-type Ca channel dihydropyridine receptor (DHPR), and sarcoplasmic reticulum Ca2+-ATPase (SERCA)]，在高膽固醇飼料餵食後的變化加以研究。此外，我們也利用免疫墨點檢驗(immunoblotting)和免疫螢光染色法，研究高膽固醇血症對細胞間聯結通道(gap junction)之主要成份蛋白 connexin 43 (Cx43)的影響 (Protocol 6)。 於研究目標3中，我們利用離體心臟灌流(Langendorff-perfused)的方式，對15隻紐西蘭白兔以di-4ANEPPS為螢光染劑進行螢光電位定位攝影檢查。其中的10隻白兔接受左冠狀動脈分枝的綁紮，以產生局部心肌缺血。我們以50%動作電位還原時間(APD50)可以心臟分為三區：Zone 1其動作電位時間均勻地縮短，Zone 3 其動作電位時間不變或略為延長，Zone 2則介於上述兩區之間其動作電位時間呈梯度變化(APD50 gradient)。針對動作電位時間restitution特性、傳導速度、心室顫動的動力學(VF dynamics)於急性局部心肌缺血發生時，在不同區(Zone 1, 2, and 3)、不同時間下的變化加以分析。同時我們也探討於急性局部心肌缺血發生時，第一型及第二型的心室顫動是否可能同時發生，以及兩者之間的交互作用對心室顫動發生的機會及心室顫動在電生理上的複雜性的影響。 結果 於研究目標1中，我們發現相較於對照組，有心室頻脈/心室顫動的心肌梗塞病患，其於急性心肌梗塞到院時的LDL-C血中濃度較高而血壓較低。追蹤三個月後，有心室頻脈/心室顫動的病患，其總膽固醇、LDL-C及三酸甘油脂之血中濃度均較高。於多變數分析後，追蹤三個月時的LDL-C(p< 0.001)血中濃度、到院時的血壓(p< 0.01)、以及急性期與追蹤三個月後的三酸甘油脂血中濃度差異(p< 0.05)，均可有效地預測心肌梗塞急性期是否會發生心室頻脈/心室顫動。心肌梗塞急性期心室頻脈/心室顫動發生的相對危險性，在LDL-C血中濃度每增加50 mg/dl的情況下是3.89(95%信賴區間為：1.74-8.69)；在急性期與追蹤三個月後的三酸甘油脂血中濃度差異每增加50 mg/dl的情況下是1.54(95%信賴區間為： 1.01-2.34)；在到院時的血壓每增加20 mmHg的情況下是0.44(95%信賴區間為：0.26-0.75)。對預測心肌梗塞急性期是否會發生心室頻脈/心室顫動而言，追蹤三個月時的LDL-C血中濃度是最顯著的獨立預測因子(independent predictor)。 於研究目標2中，Protocol 1中的血中膽固醇濃度在高膽固醇組為2097 ± 288　mg/dl，而在對照組為59 ± 9 mg/dl。而在Protocol 2中的血中膽固醇濃度在高膽固醇組則為1889 ± 577 mg/dl，而在對照組為50 ± 21 mg/dl。在Protocol 4和5中，高膽固醇組的血中膽固醇濃度也顯著地高於對照組(3022 ± 158 mg/dl vs. 42 ± 4 mg/dl ; p < 0.001 )。在Protocol 1中我們發現在高膽固醇組GAP 43陽性染色的神經密度(5587 ± 3747 vs. 2165 ± 1443 ; p < 0.001)及TH 陽性染色的神經密度(2608 ± 2592 vs. 462 ± 687 ; p < 0.001)均較對照組顯著地增加。而在Protocol 3中，研究結果顯示GAP 43陽性染色的神經密度(R2= 0.94; p<0.001)及TH陽性染色的神經密度(R2= 0.91; p<0.001)均和血中膽固醇濃度呈強烈的正相關。而在活體心臟的123I-MIBG攝影結果也顯示相較於對照組，在高膽固醇組有較高的心臟/縱膈腔顯形比例(heart/mediastinum(H/M) ratio)。同時在Protocol 1和2中我們也發現相較於對照組，高膽固醇組有較長的QTc間隔(311 ± 10 ms vs. 287 ± 9 ms, p=0.001)、較大的QTc disperion (63.5 ± 13 ms vs. 18.5 ± 4 ms, p<0.001)、較長的動作電位時間(於電刺激週期為400 ms時;APD80：193 ± 21 ms vs. 174 ± 17 ms; p<0.05)、以及較大的再極化時序差異(repolarization heterogeneity；於電刺激週期為400 ms時;標準偏差(SD)：8.7 ± 2.9 ms vs. 5.5 ± 2.0 ms, p<0.01;差值(difference)： 44.7 ± 16.9 ms vs. 25.1 ± 7.3 ms, p<0.01)。而高膽固醇組的傳導速度也顯著地較對照組降低(於電刺激週期為300 ms時;52.88 ± 1.45 cm/s versus 59.11 ± 1.64 cm/s, P<0.001)。在Protocol 2中，心室顫動自發或誘發的比例在高膽固醇組為9/12，在對照組為2/10。而在鈣離子調控方面，高膽固醇組心肌細胞的最大鈣離子流密度顯著地增加(14.0 ± 3.1 pA/pF vs. 9.1±3.4 pA/pF, p<0.01)，西方墨點檢驗則顯示高膽固醇組之蛋白SERCA的表現較對照組為低(p<0.05)，而蛋白NCX則較對照組為高(p<0.05)，但蛋白DHPR之表現在兩組之間並沒有顯著的差異。在Protocol 6中，免疫墨點檢驗則顯示心臟蛋白Cx43的表現在高膽固醇飼料餵食3週後即減少21%，而於餵食12週後則減少達60%(p<0.001)。另外，免疫螢光染色結果也顯示高膽固醇組心臟之Cx43陽性染色班塊之大小、密度也都較對照組顯著地減少，在停止高膽固醇飼料18過後，Cx43的表現可恢復。 於研究目標3中，局部心肌缺血造成缺血區域內(Zone 1)其動作電位時間restitution斜率平緩(lowest max. slope; baseline: 1.01 ± 0.52, 30 min of ischemia: 0.56 ± 0.32, 60 min of ischemia：0.55±0.30; p<0.01)，傳導速度減慢，形成了引起第二型心室顫動的條件。而在非缺血區域內(Zone 3)其動作電位時間restitution斜率更為陡峭(highest max. slope; baseline：1.65 ± 0.70, 30 min of ischemia：2.35 ± 0.71, 60 min of ischemia：2.59 ± 1.04; p<0.01)，傳導速度正常，形成了引起第一型心室顫動的條件。在引發的心室顫動中，其傅立葉主要頻譜(Dominant frequency)於Zone 2和3在局部缺血開始後隨著時間而增進，而Zone 1的主要頻譜則於局部缺血開始後先減少然後略為增加，但一般而言，Zone 1的主要頻譜仍較Zone 3為低。我們也對急性局部心肌缺血時，心室顫動電生理電波於各區域間的傳導加以分析，其結果顯示從缺血區域傳播至非缺血區的電波佔22%，從非缺血區域傳播至缺血區的電波佔41%，而約有37%的電波在兩區之間交會。同時，急性局部心肌缺血時，心室顫動的wavebreak 數目在三個區域內都會較沒有缺血時增加 (baseline, 4.3 ± 1.5; 30 min 11.7 ± 5.6; 60 min 15.6 ± 11/frame; p<0.01)。 結論 在臨床的急性心肌梗塞的情況下，我們的研究顯示血脂異常會增加心肌梗塞病患發生致命性心律不整的危險性。我們認為血中的膽固醇濃度和心肌梗塞急性期時三酸甘油酯的變化均可能在心律不整的致病機轉上扮演一定之角色。在高血脂兔動物模式中，無論是在免疫細胞化學染色的離體研究或是在123I-MIBG攝影的活體研究，我們均可發現高膽固醇血症會造成心臟的神經新生及交感神經活性增加。同時，高血脂兔也造成心臟鈣離子的調控異常(鈣離子流增加、蛋白SERCA 減少、蛋白NCX增加)和細胞間聯結通道中蛋白Cx43的減少。這些神經及電生理再塑的結果導致心臟動作電位時間延長、QTc增加、再極化時序差異變大、傳導速度變慢、進而使心室顫動發生的危險性增加。在局部心肌缺血的動物模式中，我們發現二種不同型態的心室顫動可以同時發生於急性局部心肌缺血時，無論是在缺血區域(心臟細胞的興奮性減少)或是非缺血區域(更陡峭的動作電位時間restitution特性)都產生使心律不整更容易發生的電生理變化，導致急性心肌梗塞時發生猝死或致命性心律不整的危險性增加。我們於高血脂兔及局部心肌缺氧之動物模式的研究發現，提供了我們在臨床研究中所觀察到血脂異常增加心室頻脈/心室顫動發生危險性的致病機轉上之重要基礎。同時，本研究的結果也部分解釋了何以在臨床試驗中會看到降血脂療法可以降低致命性心律不整發生率的原因。 儘管近年來在心臟血管醫學上有著長足的進步，但心臟猝死在公共衛生上仍是一個主要問題。除了心臟結構上及電生理上的再塑外，神經再塑在心臟猝死的致病機轉上也扮演一定之角色。乙狀腎上腺拮抗劑可以降低心律不整的致病機轉並不十分清楚。同樣地, 降血脂藥物(statin)顯著的抗心律不整作用也是一個明顯的難題。反觀以離子通道為作用標的的抗心律不整藥物，大多無法改善病人的預後，甚至有不良的結果。這似乎顯示自律神經的調控在臨床控制處理病人的心律上，可能成為一個新的而且有效的標的。我們的研究結果和近期一些其他的學者的研究也都支持這樣的觀點。由於血脂異常是公共衛生上的重要議題，我們有必要在將來對血脂異常所造成的神經與電生理再塑做更進一步的研究，我們將在不同的動物模式下繼續探討血脂異常引起神經再塑的機制，以及神經與電生理再塑兩者之間的交互作用在心律不整發生的致病機轉上等重要性，希望能藉此使我們對心臟猝死有更多的了解，也希望調控心臟神經再塑能成為未來臨床治療心律不整的一個新方向。Background Hypercholesterolemia (HC) is a major threat to public health in modern society and has been considered as a primary risk factor for cardiovascular diseases. Lipid-lowering interventions have been shown to reduce coronary events and all-cause mortality. It is possible that some of the beneficial effects of lipid-lowering therapy can be attributed to the reduction of ventricular tachycardia/fibrillation (VT/VF) and sudden death. Mitchell et al sought to evaluate the anti-arrhythmic effects of lipid-lowering therapy in patients enrolled in Antiarrhythmics Versus Implantable Defibrillators (AVID) trial with larger patient population. In patients with atherosclerotic heart disease (ASHD) and implantable cardiovertor defibrillator (ICD), the use of lipid-lowering drug therapy was associated with a 40% reduction in the relative hazard for VT/VF recurrence. However, it is still unclear why these lipid-lowering agents should act on the arrhythmic substrate. Tissue heterogeneity, exacerbated by electrical and structural remodeling from cardiac disease, has traditionally been considered the major factor promoting wavebreak and its degeneration to fibrillation. Recently, however, dynamic factors have also been recognized to play a key role. Dynamic factors refer to cellular properties of the cardiac action potential and calcium cycling, which dynamically generate wave instability and wavebreak. Electrical dynamic factors include restitution of action potential duration (APD) and conduction velocity (CV), short-term cardiac memory, and electrotonic currents. Calcium dynamic factors are related to dynamic calcium cycling properties. They act synergistically, as well as with tissue heterogeneity, to promote wavebreak and fibrillation. In addition to electrical remodeling, Chen’s group performed several studies to determine the relationship between nerve sprouting and cardiac arrhythmia. It is clear that, in addition to the electrical, structural, and mechanical remodeling documented in numerous models of atrial and ventricular arrhythmias, one has to consider neural remodeling. The neurogenic factors of the arrhythmia seemed to play as important a role as myogenic factors. The majority of sudden deaths are attributed to VF, often triggered by acute ischemic events. Primary VF in acute myocardial infarction predicted higher short-term mortality. Successful resuscitation in this situation is extremely difficult, resulting in a dismal survival rate. The mechanisms by which the usual cardiopulmonary resuscitation efforts fail in patients with acute regional ischemia are not well understood. Clarifying the mechanisms by which VF occurs during acute ischemia should be an important step towards more efficient prevention and/or treatment of sudden cardiac death. It is well known that myocardial ischemia alters the regional electrophysiological properties by depolarizing the cellular membrane, reducing membrane excitability, shortening APD, slowing CV, and prolonging the refractoriness beyond repolarization. Both APD restitution and excitability are important in the maintenance of VF. Prior study has classified VF into two types. The type 1 VF is associated with a steep APDR, normal excitability and multiple wavelets. The type 2 VF is associated with a flat APD restitution, low excitability, broad CV restitution, and spatiotemporal periodicity with intermittent wavebreaks. During acute regional ischemia, the ischemic region produced by acute occlusion of coronary artery is characterized by both flattening of APDR and reduced excitability, while the non-ischemic region retains normal excitability and steep APDR. Thus, both types of VF may co-exist during early phase of regional ischemia, resulting in increased difficulties in electrical defibrillation and resuscitation. Regional ischemic model can demonstrate the alterations in the ischemic and adjacent non-ischemic zone simultaneously and can help us to understand the interaction between the ischemic and non-ischemic cardiac tissue, which should be important for clarifying the mechanism of sudden cardiac death. The broad and long-term objective of this research project is to test the hypothesis that dyslipidemia increases the susceptibility of life-threatening arrhythmia underlies the mechanisms of cardiac arrhythmogenesis and sudden cardiac death in HC patients. In the traditional view, HC could result in atherosclerosis, endothelial dysfunction and plaque instability in coronary arteries, predisposing myocardial ischemia and/or infarction. It provides both triggers and substrate (tissue heterogeneity) for cardiac arrhythmia. The importance of interaction between ischemic and non-ischemic regions in initiation and maintenance of VF should be further explored. We also think that hypercholesterolemia could have direct effects on the myocardium. We further hypothesized that the interaction between neural remodeling (nerve sprouting) and electrical remodeling of myocardium induced by hypercholesterolemia underlies the mechanisms of arrhythmogenesis. In addition to coronary arteries, myocardium also severed as a target organ of serum cholesterol. Myocardial ischemia, neural remodeling and electrophysiological remodeling all contribute to the pro-arrhythmic effects of hypercholesterolemia. Meanwhile, the clinical significance of dyslipidemia on ventricular arrhythmia will also be evaluated in the clinical setting of acute coronary syndrome. The Specific Aims of this research project are as follows: Specific Aim 1 is to test the hypothesis that dyslipidemia increased the risk of developing ventricular tachyarrhythmia in the clinical setting of acute myocardial infarction. Specific Aim 2 is to test the hypothesis that HC can induce proarrhythmic neural and electrophysiological remodeling in the heart, increasing the susceptibility of VF. Specific Aim 3 is to test the hypothesis that regional ischemic tissue could result in co-existing 2 type of VF in a heart, increasing vulnerability and complexity of VF during acute coronary occlusion. Materials and methods In Specific Aim 1, a total of 58 patients experiencing VT/VF within 24 hours after the onset of MI were matched to 58 patients with MI but without VT/VF. Sex (104 males), age (58 ± 10 years), and the use of thrombolytic therapy (n=82) were matched in both groups initially. The lipid profiles including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride were measured during the first week and at the 3rd month after the index MI. Other coronary risk factors, clinical, hemodynamic, and angiographic characteristics were also included in our assessment. In Specific Aim 2, We fed rabbits with either high cholesterol (HC, N=10, 6, 15, 25) or standard (S, N=10, 6, 15, 20) chows for 12 weeks (Protocol 1,4-6), and with HC (N=12) or S (N=10) chows for 8 weeks (Protocol 2). In Protocol 3, 10 rabbits were fed with various protocols to observe the effects of different serum cholesterol levels. Twelve-lead electrocardiograms were recorded for QTc and QTc dispersion analysis very 2 weeks during the feeding period in Protocol 1. Electrophysiological study and optical mapping of Langendorff- perfusion hearts were performed at the end of feeding period in Protocol 1 and 2. The alterations in APD, APD dispersion, CV and vulnerability of VF were studied. We used immunocytochemical stain of GAP-43 (a maker for nerve sprouting) and TH (a maker of sympathetic nerve) to evaluated sympathetic activity “in vitro” in Protocol 1 and 3; and used myocardial 123I-meta- iodobenzylguanidine (123I-MIBG) imaging to evaluated sympathetic activity “in vivo” in Protocol 4. Whole-cell clamp recordings of calcium inward currents and protein expression of Na+/Ca2+ exchanger (NCX) L-type Ca channel dihydropyridine receptor (DHPR), and sarcoplasmic reticulum Ca2+-ATPase (SERCA) were studied to explore alterations in calcium handling in HC rabbit hearts in Protocol 5. Immunoblotting and immuofluorescence microscopic study of connexin 43 (Cx43) were also done to see the remodeling of gap junction in HC rabbits hearts in Protocol 6. In Specific Aim 3, optical mapping studies were performed with di-4ANEPPS in 15 Langendorff-perfused rabbit hearts. Branches of coronary artery were ligated to create regional ischemia in 10 hearts. APD measured to 50% repolarization (APD50) during ischemia showed an area with uniformly shortened APD50 (Zone 1), normal or lengthened APD50 (Zone 3) and an area in between with an APD50 gradient (Zone 2). APD restitution, CV and VF dynamics were analyzed in different areas (Zone 1, 2, and 3) of regional ischemic hearts. We further determined whether Type I and Type II VF could be demonstrated in heart with regional ischemia and the importance of interaction between 2 types of VF on VF complexity. Results: In Specific Aim 1, patients with VT/VF had a higher level of LDL-C and a lower blood pressure on the initial arrival during the acute myocardial infaction. At the 3-month follow up, those patients with VT/VF had a higher level of TC, LDL-C and triglyceride. In multivariate analysis, the LDL-C (p< 0.001) at the 3-month follow up, the mean blood pressure on arrival (p< 0.01), and the difference in the triglyceride levels between the first week and the third month (p< 0.05) were all the independent predictors for the occurrence of VT/VF in the acute stage of MI. The relative risk of occurrence of VT/VF was 3.89 for each increasing in 50 mg/dl of LDL cholesterol (95% CI: 1.74-8.69), 1.54 for each increasing in 50 mg/dl of the difference between 1-week and 3-month triglyceride (95% CI: 1.01-2.34), and 0.44 for each increasing in 20 mmHg of mean blood pressure (95% CI: 0.26-0.75). The 3-month serum LDL cholesterol was the most significant independent predictor of the occurrence of VT/VF during the first day of acute MI. In Specific Aim 2, the serum cholesterol levels were 2097 ± 288 mg/dl in HC group and 59 ± 9 mg/dl in S group for Protocol 1 and were 1889 ± 577 mg/dl in HC group and 50 ± 21 mg/dl in S group for Protocol 2. In Protocol 4 and 5, the serum cholesterol (3022 ±158 mg/dl vs. 42 ±4 mg/dl; p<0.001) levels were also significantly higher in HC group by the end of 3-month period. Density of GAP43- and TH-positive nerves in the heart were significantly higher in HC (5587 ± 3747 and 2608 ± 2592 μm2/mm2) than S (2165 ± 1443 and 462 ± 687μm2/mm2, respectively, p<0.001) in Protocol 1. Protocol 3 showed a strong correlation between serum cholesterol level and nerve density for GAP43 (R2= 0.94; p<0.001) and TH (R2= 0.91; p<0.001). The cardiac MIBG uptake of HC group, which was evaluated with heart/ mediastinum (H/M) ratio, was higher (3.39 ± 0.11 vs. 2.90 ± 0.10; p<0.05) than that of the age-matched control group. Compared with S, HC rabbits had longer QTc intervals (311 ±10 ms vs. 287 ± 9 ms, p=0.001), more QTc dispersion (63.5 ± 13 ms vs. 18.5 ± 4 ms, p<0.001), longer APD (At pacing cycle length, PCL=400 ms;APD80: 193 ± 21 ms vs. 174 ± 17 ms; p<0.05) and increased heterogeneity of repolarization (At PCL=400 ms; SD: 8.7 ± 2.9 ms vs. 5.5 ± 2.0 ms, p<0.01; difference: 44.7 ± 16.9 ms vs. 25.1 ± 7.3 ms, p<0.01) in Protocol 1 and 2. The measured CVs for HC rabbits were also significantly less than that of the control (At PCL=300 ms; 52.88 ± 1.45 cm/s versus 59.11±1.64 cm/s, P<0.001). Ventricular fibrillation was either induced or occurred spontaneously in 9/12 of hearts of HC group and 2/10 of hearts in S group in Protocol 2. The peak L-type calcium inward current density was significantly higher in HC myocytes (14.0 ± 3.1 pA/pF vs. 9.1 ± 3.4 pA/pF, p<0.01). Western blot experiments revealed that protein expression of SERCA in the HC strips decreased (p<0.05), but that of the NCX increased (p<0.05) in Protocol 5. The protein expression of the DHPR was similar between these two groups. Western blotting demonstrated a 21% reduction of connexin43 (Cx43) protein expression within three weeks. Along the feeding protocol for 12 weeks, expression of cardiomyocyte Cx43 protein progressively decreased (60% reduction at 12 weeks, P<0.001) in Protocol 6. Such a reduction was also demonstrated by immunoconfocal microscopy with decreases in the proportion of total tissue area occupied by Cx43 signals and the size/density of Cx43 gap-junctional plaques. Withdrawing a cholesterol- enriched diet for 18 weeks restored the Cx43 protein expression. In Specific Aim 3, ischemia flattened APD restitution slope (lowest max. slope; baseline: 1.01 ± 0.52, 30 min of ischemia: 0.56 ± 0.32, 60 min of ischemia: 0.55±0.30; p<0.01) and reduced conduction velocity in ischemic zone (Zone 1), creating a condition for type 2 VF. APD restitution steepened (highest max. slope; baseline: 1.65 ± 0.70, 30 min of ischemia: 2.35 ± 0.71, 60 min of ischemia: 2.59 ± 1.04; p<0.01) and the conduction velocity changed little in the non-ischmic zone (Zone 3), creating a condition for type 1 VF. During induced VF, the dominant frequency in Zones 2 and 3 progressively increased after the onset of ischemia. The dominant frequency in Zone 1 (ischemic zone) first decreased and then slightly increased, but typically remained less than the dominant frequency in the Zone 3. We also analyzed the spread of activation into and out of the ischemic region by mapping activation sequences in those VF episodes at 30 min of ischemia. We found about 22% of wavefronts propagating from ischemic zone to non-ischemic zone, 41% of wavefronts propagating from non-ischemic zone to ischemic zone, and 37% of waves collided near the ischemic border zone. The number of wavebreaks increased with time in all 3 zones (baseline, 4.3 ± 1.5; 30 min 11.7 ± 5.6; 60 min 15.6 ± 11/frame; p<0.01). Conclusion: In the clinical setting of acute myocardial infarction, our study suggests that dyslipidemia imposes a higher risk of developing tachyarrhythmia in the acute phase of myocardial infarction. It is possible that the baseline serum lipid and the triglyceride levels during acute phase of myocardial infarction played a significant role in the arrhythmogenesis. In the rabbit model of HC, HC induced significant nerve sprouting and sympathetic hyperinnervation in both immunocytochemical studies “in vitro” and 123I -MIBG imaging “in vivo”. Abnormal calcium handling (increased ICa, decreased SERCA protein expression, and increased NCX protein level) and down-regulation of connexin 43 were also demonstrated in heart with HC. The neural and electrophysiological remodeling was associated with prolonged APD, longer QTc intervals, increased repolarization dispersion, decreased CV and increased ventricular vulnerability to fibrillation. In regional ischemic rabbit heart model, two types of VF can co-exist during acute regional ischemia. Both ischemic (decreased excitability) and non-ischemic regions (steepen APD restitution) developed proarrhythmic changes during regional ischemia, therefore, contributing to the increased ventricular vulnerability to VF and sudden death during acute coronary occlusion. The findings in both HC rabbit model and regional ischemic model provided mechanistic insight into the dyslipidemia predisposing to the occurrence of VT/VF during acute stage of myocardial infarction. Meanwhile, these results may also partially explain the beneficial effects of lipid-lowering drugs in reducing the risk of sudden cardiac death in large-scale clinical trials. In spite of recent advances in cardiovascular medicine, sudden cardiac death remains a major public health problem. In addition to anatomical and electrical remodeling, neural remodeling also plays a role in the generation of sudden cardiac death. The mechanism by which β-blocker reduces the risk of arrhythmia has been unclear. Likewise, the dramatic antiarrhythmic effect of statin remains an outstanding puzzle. In contrast to the poor outcome of drugs targeting ion channel function, it appears that autonomic control may become a new and effective target for the management of cardiac rhythm. Such a view is supported by a large body of recent evidence showing that neural remodeling as manifested by sympathetic hyperinnervation and nerve sprouting exists in diseased hearts. Due to the significant health implications of HC in humans, it is necessary to further study the effects of HC induced neural and electrical remodeling on potential risk of cardiac arrhythmias. We will conduct future studies to study the interaction between electrical remodeling and neural remodeling using different animal models. The additional knowledge obtained from future project will further extend the understanding of the mechanisms of sudden cardiac death, and potentially lead to new therapeutic approaches for the management of heart rhythm.1 中文摘要 3 2 緒論 9 2.1 血脂異常與死亡率 9 2.2 交感神經活性與心臟猝死: 致心律不整之神經再塑 10 2.3 交感神經功能之評估 12 2.4 電生理動力學與心臟組織異質性之交互作用: 致心律不整之電生理再塑 14 2.5 電生理再塑之功能性評估: 螢光電位定位攝影 17 2.6 局部心肌缺氧的重要性 18 2.7 鈣離子調控與心律不整 19 2.8 本論文之研究假說與研究目的 20 3 研究設計與方法 22 3.1 臨床心肌梗塞的病患其血脂異常與致命性心室心律不整關係之探討 22 3.2 高膽固醇血症所引發的心臟的神經和電生理再塑，及以與心室顫動發生關連之探討 24 3.3 急性局部心肌缺血時，心室顫動在電生理上的複雜性之探討 30 4 研究結果 32 4.1 臨床心肌梗塞的病患其血脂異常與致命性心室心律不整關係之探討 32 4.2 高膽固醇血症所引發的心臟的神經和電生理再塑，及以與心室顫動發生關連之探討 33 4.3 急性局部心肌缺血時，心室顫動在電生理上的複雜性之探討 37 5 討論 40 5.1 血脂異常增加心肌梗塞的病患發生心室心律不整的危險性 40 5.1.1 高血脂症和心室心律不整 40 5.1.2 三酸甘油脂的急性變化和心室心律不整 40 5.1.3 研究限制 41 5.2 高膽固醇血症所引發致使心室顫動更易發生之心臟神經和電生理再塑 42 5.2.1 高膽固醇兔發生神經芽新生的可能機轉 42 5.2.2 神經芽新生和心肌肥厚 43 5.2.3 高膽固醇血症和電生理再塑 43 5.2.4 高膽固醇血症和鈣離子調控異常 44 5.2.5 高膽固醇血症和細胞間聯結通道Cx43蛋白的表現量的減少 45 5.2.6 心臟神經再塑和電生理再塑之交互作用 46 5.2.7 高膽固醇血症增加發致使心室顫動更易發生的可能機轉 47 5.2.8 研究限制 48 5.3 急性局部心肌缺血時，第一型與第二型的心室顫動可同時發生並增加心室顫動在電生理上的複雜性 48 5.3.1 急性局部心肌缺血時心室顫動 wavebreak 增加的可能機轉 48 5.3.2 急性局部心肌缺血時，非缺血心臟組織發生的電生理特性改變的可能機轉 50 5.3.3 臨床上的可能應用 50 5.3.4 研究限制 51 6 展望 52 7 論文英文簡述 54 8 參考文獻 61 9 圖表 79 10 附錄： 104 10.1 博士論文英文版 104 10.2 博士班修業期間發表相關論文 14910841768 bytesapplication/pdfen-US心律不整血脂異常神經系統急性心肌梗塞鈣離子調控細胞聯結通道arrhythmiadyslipidemianerve systemacute myocardial infactioncalcium handlinggap junction[SDGs]SDG3texthttp://ntur.lib.ntu.edu.tw/bitstream/246246/55532/1/ntu-94-D87421004-1.pdf