小電導鈣激活鉀通道與心室心律不整之探討

Small-conductance Ca2+-activated K+ Channels in Ventricular Arrhythmia

游治節(Chih-Chieh Yu)

指導教授 ： 賴凌平 ； 楊偉勛

國立臺灣大學/醫學院/臨床醫學研究所/博士(2016年)

https://doi.org/10.6342/NTU201603636

摘要

小電導鈣激活鉀離子電流是一種受細胞內鈣離子濃度調控的離子電流。它最先被發現在中樞神經系統，負責神經元可刺激性的調控 (afterhyperpolarization)。之後發現在心房細胞中占有重要的地位，也影響傳導系統的自發性(automaticity)及傳導。最近，一些動物實驗研究發現小電導鈣激活鉀離子電流在心臟衰竭或梗塞的心肌細胞會上揚，可能和心室頻脈有關。
為了進一步測試小電導鈣激活鉀離子電流在人類心臟細胞的角色，我們首先使用patch clamp 的技術來測試小電導鈣激活鉀離子電流的阻斷劑，apamin，的專一性。我們用高劑量的apamin（500 nM）測試所有主要的人類心臟離子電流，包括L 型鈣，鈉和主要的鉀電流（包括IKs 通道，IKr，IK1，Ito），都沒有發現顯著的抑制或促進。於是，我們便放心的使用apamin 來檢測人類為了心臟移植而取出的衰竭心臟進行光學標測研究(optical mapping)。我們發現不管在心內膜、中層心肌或心外膜，在注射apamin 後膜動作電位持續時間(action potential duration)都有顯著的增加。此外也在衰竭心臟的心肌內觀察到M 細胞島(M cell islands)，在這島裡面，小電導鈣激活鉀離子電流較周邊組織少，而注射apamin後，可觀察到膜電位不穩定的現象(electric alternans)。有趣的是，我們還發現apamin 會顯著減低傳導速度，這意味著小電導鈣激活鉀離子電流可能參與心肌細胞的傳導，而心肌組織的免疫染色也發現小電導鈣激活鉀離子通道蛋白大量存在於細胞間的intercalated discs，呼應小電導鈣激活鉀離子電流可能參予心肌細胞傳導的角色。然而，由於小電導鈣激活鉀離子電流上揚而縮短動作電位持續時間的同時，卻惡化動作電位和鈣電流持續時間之間的差異，而這差異的大小被認為是心律不整是否容易發生的關鍵。為了進一步探討小電導鈣激活鉀離子電流與人類心室心律不整的關係，我們進行一個臨床研究，將臨床上因為曾經發生心室頻脈而須植入體內去顫器的病患，測試其KCNN2 基因多型性(polymorphism)是否與正常人有不同的地方，結果顯示，確實有兩個KCNN2 基因多型性的核苷酸序列，與心室頻脈的發生有密切相關，帶有此序列的人大約會增加2.6〜2.9 倍心室頻脈的風險，證實了人類小電導鈣激活鉀離子電流與心室頻脈的關聯。
小電導鈣激活鉀離子電流在許多疾病的狀態下會因為細胞內鈣離子增加而上揚，進而縮短膜動作電位持續時間，產生類似保護的作用，例如心臟衰竭、QT 過長症候群、還有像最近興起的調鈣激素症候群(calmodulinopathy)。我們用patch clamp 的技術來測試小電導鈣激活鉀離子電流是否會受到這些突變的調鈣激素影響，結果顯示這些突變的調鈣激素確實減弱了小電導鈣激活鉀離子電流受鈣離子調控的能力。
我們利用人體心臟組織以及血液關於小電導鈣激活鉀離子電流相關的研究已經進一步證實過去在動物實驗中看到小電導鈣激活鉀離子電流與心室頻脈的相關性也在人類心臟出現。在這些研究中，我們也觀察到一些新的發現，例如小電導鈣激活鉀離子電流可能影響心臟肌肉組織的傳導速度，這些研究的結果需要進一步研究來證實並探討背後的詳細機制，例如小電導鈣激活鉀離子電流與心肌傳導速度的關係、KCNN2 基因多型性如何影響心臟猝死的風險。在研究小電導鈣激活鉀離子電流的這條道路上，我們已經又向前邁進一大步，但距離我們想要真的能利用小電導鈣激活鉀離子電流做為治療或預防心因性猝死的目標還很遠，仍待更多科學家共同的努力及付出。

關鍵字

小電導鈣激活鉀通道 ； 心室心律不整

並列摘要

Small conductance Ca2+-activated K+ (SK) current is a repolarization current gated by increased intracellular calcium concentration. It was firstly found abundant in the
central nervous system and was responsible for afterhyperpolarization of the neurons. It was later found to be important in the repolarization of atrial myocytes and play important roles in automaticity and atrioventricular node conduction. Recently, some animal studies showed SK current was found to be upregulated in diseased heart ventricles, and was involved in arrhythmogenesis.

To further test the role of SK current in human ventricles, we first used patch clamp technique to test the specificity of SK current blocker, apamin. We tested all major human cardiac ion currents, including L-type Ca2+, Na+ and the major K+ currents (IKs, IKr, IK1, Ito) with high dose apamin (500 nM) and found no significant effects. We then used apamin for the following human explanted failing heart optical mapping study. We took left ventricle free wall wedge, infused with calcium-sensitive and voltage-sensitive dyes. We used high resolution camera to capture the optical change of the dyes before and after apamin. We found a significant increase of action potential duration after apamin, especially at high pacing cycle length at all three layers of endocardium, midmyocardium and epicardium. In M cell islands, less increase of SK current was observed, and electrical alternans was observed after apamin. Interestingly, we also found a significant reduction of conduction velocity after apamin, implying that SK current was involved in conduction. This observation was also strengthened by our immunostaining study of the wedge that SK protein was abundant in the intercalated discs. However, because upregulation of SK current shortened action potential duration, it enhanced the discrepancy of action potential duration and calcium transient duration, which has been proposed as a pro-arrhythmic character. We further tested the association between SK current and human arrhythmia by conducting a genetic association study between KCNN2 and human sudden cardiac death. We collected the blood sample of the patients with history of aborted sudden cardiac death due to ventricular tachyarrhythmias. We tested the association of tachyarrhythmias and KCNN2 polymorphisms and found 2 strongly associated tag single nucleotide polymorphisms (SNPs). The odds ratio was around 2.6~2.9, proving the involvement of SK current in human ventricular tachyarrhythmias.

SK current acts like a protective current when the myocardium is exposed in a condition that may increase intracellular calcium, such as heart failure, long QT syndrome and the recently rising syndrome, calmodulinopathy. We used patch clamp technique to test those human arrhythmogenic mutant calmodulin on SK current and found a failure of the mutant calmodulin to fully open SK2 channels in response to elevated intracellular calcium.

Our studies in human tissues further strengthened the observation in animal studies that SK current was involved in ventricular arrhythmias when the heart was exposed in diseased status. In these studies, we also had some very novel findings that needed further studies to confirm and explore the mechanisms, such as involvement of SK current in conduction and the increased risk of sudden cardiac death when carrying KCNN2 polymorphisms. We’ve moved a step forward on the way of understanding SK current, but more effort is warranted until we achieve the goal of using SK current as the target to prevent sudden cardiac death.

並列關鍵字

ventricular fibrillation ； small-conductance calcium-activated potassium current ； calcium current ； action potential duration

參考文獻

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