2015-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/656366摘要：心室頻脈引起之心因性猝死是最嚴重的一種心臟病，目前已知電生理特性的變化和心因性猝死有關，尤其在心衰竭的病人。但是目前對於為何有些心衰竭病人會心因性猝死，有些卻不會，目前並不清楚。另外，對於為什麼在心衰竭心臟收縮功能變差後，就容易產生心律不整或心室頻脈的機制，目前也不清楚。在心衰竭病人，目前所知由於內質網鈣離子幫浦表現減少，心臟鈣離子在心收縮及舒張週期的動態生理會有異常，鈣離子在舒張期回收至內質網的過程變慢，而導致內質網內鈣離子濃度下降以及收縮功能變差。至於此病態生理和心律不整的關係，目前並不清楚，因此，了解兩者的關係將有助於找出心衰竭病人易罹患心因性猝死之高危險族群及對治療心室頻脈引起之心因性猝死也有很大的幫助。最近研究指出心臟在每一間隔心跳之間去極化或動作電位的交替變化，會反應在心電圖T 波型態之交替變化，這種現象會發生在正常心臟心跳很快時或是在內質網鈣離子幫浦表現較少之區域(如心內層)，如此會造成在空間上不同區域間不反應期不整，有助於傳導阻斷的發生而引發再傳入性迴路心律不整，此現象被認為和會不會發生心室性心律不整或猝死有密切相關。因此研究心衰竭心臟這種去極化交替變化的機制，將有助於了解為何心衰竭病人容易罹患心律不整的原因。目前為止，對於去極化交替變化之機轉研究，都局限在正常的心肌，而在心衰竭心臟去極化交替變化在心律不整或心室頻脈機轉之角色，目前研究不多。因此本三年計畫的研究假設為：心衰竭病人由於內質網鈣離子幫浦表現減少而導致鈣離子在心週期動態生理異常，進而產生收縮功能變差以及去極化或動作電位期間的交替變化，引起傳導阻斷及產生心室性心律不整，這樣的假設是根據:(1)目前已知心衰竭病人內質網鈣離子幫浦表現減少而造成心臟鈣離子在心週期動態生理會有異常；(2)心臟鈣離子心週期動態生理異常在正常心臟已被證實和心臟去極化交替變化及心律不整機轉有密切關係。要證實這一假設最佳的方式為嘗試在心衰竭動物模式利用基因轉殖方式過度表現內質網鈣離子幫浦，來改正心衰竭內質網鈣離子幫浦表現減少的狀態，以證實是否心衰竭易發生心臟去極化交替變化的病態生理同時獲得改善。因此在此三年的計畫中，我們將嘗試利用斑馬魚心衰竭動物模式，來探討這個問題，使用斑馬魚模式是因為斑馬魚在基因轉殖或改變的方法較方便也確定可行，另外斑馬魚電生理特性和人類極為相似。在第一年我們將建立同時鈣離子及動作電位之即時螢光記錄系統來研究斑馬魚心臟去極化交替變化等電生理特性。在第二年我們將利用Morpholino 基因剔除方式建立斑馬魚心衰竭模式，且證實在心衰竭斑馬魚會有內質網鈣離子幫浦表現減少，鈣離子心週期動態生理失衡及心臟去極化交替變化的病態生理；在第三年我們將在心衰竭斑馬魚心臟過度表現內質網鈣離子幫浦以嘗試治療修正心衰竭內質網鈣離子幫浦表現減少，以期能改善心衰竭易產生心臟去極化交替變化的病態生理，如此將可證明針對鈣離子生理的基因治療，可以改善心衰竭心臟去極化交替變化病態生理及治療心律不整。總結而言，本研究計畫以新穎的動物模式來探討一項重要的臨床問題。<br> Abstract: Sudden cardiac death (SCD) is the most devastating manifestation of heart disease. Theelectrophysiological changes that predispose to SCD in systolic heart failure (HF) have beenstudied extensively. However, little is known of the complex sequence of events that incitemalignant arrhythmias in some patients but not in others. Furthermore, the mechanismslinking mechanical to electrophysiological dysfunction in HF are unclear. Impaired calcium(Ca) cycling, specifically, impaired Ca reuptake due to reduced expression of sarcoplasmicreticulum Ca ATPase (SERCA2a), is the most striking abnormality of failing myocytes, andis responsible for contractile dysfunction in HF. It remains unclear how this influencessusceptibility to ventricular arrhythmias or SCD. Improved understanding of thefundamental relationships between mechanical and electrophysiological cardiac dysfunctionis critical for developing better approaches for identifying and treating patients at high riskfor SCD. It has been recognized that beat-to-beat alternation of cardiac repolarization oraction potential duration, manifested clinically as subtle T wave alternans (T-ALT), is ahighly sensitive marker of susceptibility to SCD. Beat-to-beat alternation of action potentialduration will produce a repolarization gradient, or spatial heterogeneity of refractoriness,which facilitates the occurrence of conduction block and the initiation of reentrantarrhythmia. Therefore, by elucidating the mechanisms of cardiac alternans in HF, there is anopportunity to understand why HF enhances susceptibility to arrhythmias. However,essentially all previous research on cardiac alternans has focused on normal hearts, ratherthan HF. Therefore, the role of cardiac alternans in the most common setting for SCD, i.e.HF, is unknown. We hypothesized that impairment Ca cycling processes which contribute tocontractile dysfunction in HF, also enhance susceptibility to cardiac alternans, that is,beat-to-beat alternation of action potential duration or repolarization, and, in turn,susceptibility to arrhythmias. This hypothesis is based on recent findings that (1) HF causesimpaired sarcoplasmic reticulum (SR) Ca reuptake due to reduced SERCA2a expression; (2)this perturbation has also been implicated as mechanisms of cardiac alternans in non-failingor normal myocytes. The most convincing way to prove this hypothesis is that restoration ofdefective Ca cycling in HF, specifically, by overexpressing cardiac SERCA2a, reverses thesusceptibility to cardiac alternans back to normal. Therefore, in this three years’ proposedproject, we will use high-resolution dual Ca and action potential mappings to evaluatecardiac alternans on a novel HF model- zebrafish, because of its easy feasibility of geneticmanipulation and similar electrophysiological properties with those of humans. In the firstyear, we will establish a dual voltage-Ca optical mapping system to record action potentialand Ca transient of the zebrafish embryonic heart, and establish the protocol to inducecardiac alternans. In the second year, the zebrafish HF model will be established bymorpholino knockdown of titin and troponin T, and defective Ca cycling along with thesusceptibility to cardiac alternans will be validated. In the third year, gene transfer targetingthe SERCA2a protein will be performed to restore the defective Ca cycling andsusceptibility to cardiac alternans. Gene transfer techniques targeting defective Ca cyclingproteins, such as SERCA2a protein, can provide a novel strategy for engineering the“alternans-resistant” heart. In conclusions, by using a novel animal model, this three years’project incorporates an innovative approach to address an important clinical issue.斑馬魚心衰竭 心因性猝死 心臟去極化交替變化zebrafishheart failuresudden cardiac deathcardiac alternans