| 摘要: | 心律不齊引起的心臟猝死(SCD)是造成台灣每年100萬個心血管疾病患者的主要死因之一，而 且佔心血管問題導致死亡有近50%的比率。對於心律不齊所引發心臟猝死的起因，我們目前尚 未完全了解。先前的研究中，我們利用HEK293細胞來過度表達與調控心律節奏有關的HCN2 與HCN4離子通道，結合電生理的方法，發現赂胺酸磷酸化(tyrosine phosphorylation)能調控HCN 離子通道的上膜量，同時HCN2能夠調控長效型鈣離子通道（LTCCs)開關的程度(gating property)。 在HEK293細胞上我們也發現了 HCN離子通道與鈣離子通道在細胞膜上的位置十分接近，進而 探討兩者在物理結構上是否有相互作用。 然而，以上實驗都屬於細胞層級(in vitro)，本計劃主要是在探討酪胺酸磷酸化在整體動物(in vivo)，是否會調控心律節奏以及引發心律不齊。根據我們2012年國科會計晝的研究，利用靜脈 注射的方式分別給予大鼠⑴酪胺酸磷酸酶抑制劑sodium orthovanadate (Na3V〇4)及(2)酪胺酸酶 抑制劑 PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine)，來增加或降低酪胺 酸磷酸化程度，藉此觀察其相關之調控機制。 酪胺酸磷酸酶抑制劑(Na3VO4)能有效抑制磷酸酶(phosphatase)，並影響HCN離子通道上的酪胺 酸磷酸化位點使上膜量增加，這個現象造成了在大鼠的心電圖(ECG)上顯示出心律不齊。結果發 現數種不同亞型於心室相關的心律不齊，因此我們研判可能與心室上的第二型HCN離子通道 (HCN2)有關。同時在ECG上我們也觀察到有long QT的現象發生，long QT的現象在先前的 研究中表明了與心室上的鈣離子通道有關，且我們先前的研究中亦提及HCN離子通道與鈣離子 通道之間的相互關係，從而進一步探討long QT是否與HCN通道及鈣離子通道的交互作用有關。 酪胺酸激酶抑制劑PP2為Src磷酸激酶(Src kinases)的抑制劑，抑制了磷酸激酶(kinase)而影響到 HCN離子通道上的酪胺酸磷酸化位點使上膜量減少，這個現象造成在大鼠的ECG上會顯示出房 室傳導阻滯(AV Block)，在此計晝中我們要探討房室傳導阻滯(AV Block)與心房上的第四型 HCN離子通道(HCN4 )是否有關。 第一年的研究我們主要是利用Hemodynamic measurements的技術在大白鼠體内以靜脈注射 方式注入Na3VO4來提升酪胺酸磷酸化的程度1)以Weston blot、Real Time PCR、Biotinylation、 Co-immunoprecipitation、Confocal image technology 來觀察 HCN2 的上膜量的程度以及酪胺酸磷 酸化程度2)以Whole cell patch clamp觀察鈣離子通道開關的程度3)觀察是否鈣離子通道與 HCN離子通道具有物理結構上的交互作用4)以Heart rate variability (HRV)觀察使用Na3V〇4造 成之HCN2通道上酪胺酸磷酸化的提升與鈣離子通道對心臟周圍的交感神經與副交感神經(尤其 是副交感神經，多數分布於心室)是否存在相互作用。 第二年的研究我們主要是利用Hemodynamic measurements的技術，同樣在大白鼠體内注入酪胺 酸酶抑制劑PP2來降低酪胺酸磷酸化的程度：1)探討以PP2降低酪胺酸磷酸化程度誘導HCN4 造成房室傳導阻滯(AV Block)背後的機制2)以Heart rate variability (HRV)觀察使用PP2造成 HCN通道上酪胺酸磷酸化的降低與鈣離子通道對心臟周圍的交感神經與副交感神經是否存在相 互作用。 本研究之成果在未來希望能以Na3VO4、PP2為基礎，提供相關心律不齊研究的初步基礎，期望 能更加瞭解調控心律之相關機制，以利控制或延緩心律不齊的發生。
Cardiac arrhythmias caused sudden cardiac death (SCD), which affects approximately 1,000,000 people of cardiovascular patients in Taiwan. It is estimated to account for nearly 50 percent of all death from cardiovascular causes. However, the underlying mechanisms of cardiac arrhythmias still remain obscure due to lack of a good animal model system with reliably inducible and controlled real-time cardiac arrhythmias. In previous studies, we employed HEK293 cells to overexpress the pacemaker Hyperpolarized Cyclic-Nucleotide gated (HCN) channels combined with electrophysiology method (such as whole cell recording) to study the regulation of HCN channel activities induced by tyrosine phosphorylation. Our former results indicated that either current densities or current kinetics of HCN channels can be modulated by tyrosine phosphorylation. Furthermore, HCN channels can modulate the inactivation of L-type calcium channels (LTCCs) in HEK293 cells, which increase the possibility of the cross talk between HCN channels and LTCCs. However, those experiments mentioned above are all in vitro. Our main purpose of this research is to investigate if/how tyrosine phosphorylation regulates the rhythm of the heartbeat and induces cardiac arrhythmias in vivo. According to our previous National Science Council (NSC) research project in 2012, we have hypothesized that tyrosine phosphorylation is capable of modulating cardiac activities in vivo. By using intravenous injection, we implied to the rats and results indicated that 1) sodium orthovanadate (Na3VO4), tyrosine phosphatase inhibitor, is able to induce different subtypes of ventricular arrhythmias and QT prolongation by increasing the tyrosine phosphorylation level. 2) 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine (PP2), Src kinases inhibitor, is capable of inducing atrial arrhythmias by reducing the tyrosine phosphorylation level. As the II subtype HCN channels (HCN2) and LTCCs dominantly express on the ventricle, they may be responsible for the abnormal ventricular rhythm as well as QT prolongation induced by different doses of Na3V〇4 administration. On the other hand, the IV subtype HCN channels (HCN4) dominantly located at the atrium, which make its contribution to atrial arrhythmogenesis induced by PP2 more likely. In this study, we would like to base on our previous results to extend into two year proposal: In first year, we will employ hemodynamic measurements and treat Na3V〇4 by intravenous (IV) injection in rats to increase tyrosine phosphorylation in order to 1) investigate the expression of total and membrane HCN2 channel proteins and tyrosine phosphorylation level in rat model by biochemistry approaches (i.e. western blot, co-immunoprecipatation, Real-Time PCR, biotinylation, and confocal image); gating properties of HCN2 channels by using whole cell recording (patch clamp) will also be employed; 2) examine the expression of total and membrane proteins of LTCCs by biochemistry approaches; whole cell recording will be used to identify its gating properties; 3) investigate the physical interaction between HCN2 channels and LTCCs; 4) identify if/how the activities of autonomic nervous systems (ANS) (i.e. sympathetic/para sympathetic nerves) will be affected by increase of tyrosine phosphorylation. Heart rate variability (HRV) with its relevant parameters will be examined. In second year, hemodynamic measurements will be employed and PP2 will be administrated to decrease tyrosine phosphorylation in rats. We are going to 1) investigate the expression of total and membrane HCN4 channel proteins and tyrosine phosphorylation level in rats by biochemistry approaches (i.e. western blot, co-immunoprecipatation, Real-Time PCR, biotinylation, and confocal image); whole cell recording will be used to identify its gating properties; 2) identify HRV with its relevant parameters to examine whether the activities of ANS. |
