Exploring the role of ATP1A3 mutations in sudden unexplained death in epilepsy

项目摘要/摘要 癫痫原因不明猝死(SUDEP)是指有癫痫病史的患者的原因不明的猝死。 以及处于合理健康状态的癫痫患者。它是癫痫患者死亡的主要原因之一，也是一种常见的 神经性死亡的全部原因。据信是由心脏和神经因素引起的，癫痫 诱发性心动过缓是SUDEP的危险因素，并可能在易感心肌引发致死性室性心律失常。 开发一个强大的实验模型来概括这一点，将为基础研究打开大门 开发关键药物疗法。SUDEP是儿童交替性偏瘫(AHC)患者的悲惨结局 它的特征是癫痫、肌张力障碍、瘫痪，特别是在心动过缓的情况下猝死。其中 AHC患者中，90%在ATP1A3编码的α-3催化亚单位存在潜在的病理遗传变异 Na/K-ATPase泵(ATP1A3)。我们已经确定了最常见的与AHC相关的 心动过缓时心电图上的ATP1A3变异(D801N)和短QT间期与室颤人类诱导多能性 来自ATP1A3-D801N阳性儿童的干细胞来源的心肌细胞(hiPSC-CMD801N)表现为缩短 复极时间、钙稳态紊乱和延迟后除极，这些都是心律失常的触发因素。 携带D801N变体(Atp1a3D801N)的敲入小鼠会癫痫发作、心动过缓和猝死。此外，Atp1a3D801N 与对照组相比，小鼠容易发生室性心律失常，特别是在心率较低的情况下。总而言之， 这些发现增加了心脏中ATP1A3的破坏是AHC患者SUDEP的基础的可能性 心动过缓引发的心律失常。我们假设D801N降低了泵浦函数，导致复极化缩短 时间和致死性室性心律失常的易感性。我们的目标是使用AHC作为模型来建立ATP1A3在 心动过缓的作用机制，探讨心动过缓的致心律失常作用。要在以下方面处理此问题 以创新和严谨的方式，我们将利用患者派生的hiPSCD801N和基于小鼠Atp1a3D801N的模型来 体外、体外和体内研究，以确定心脏心律失常易感性的机制。具体来说，我们 提出1)D801N引起心肌细胞动作电位时程缩短的机制，2) D801N诱发心律失常的三维组织模型和体外分析机制 癫痫发作引起的心动过缓是Atp1a3D801N小鼠“脆弱”心肌的致心律失常诱因。在……里面 为了实现这些目标和总体目标，我们将确定ATP1A3在心血管生理学中的功能和它的 在心脏复极、钙信号和室性心律失常中的作用，从而识别分子靶点 药物疗法。最后，这将开发稳健和严格的模型来确定猝死的机制。 AHC，并将更广泛地提供对SUDEP机制的见解。

PROJECT SUMMARY/ABSTRACT Sudden unexplained death in epilepsy (SUDEP) is the sudden and unexplained death of a patient with a history of seizures and epilepsy who is in a reasonable state of health. It is a major cause of death in patients with epilepsy and a common cause of neurologic death overall. Believed to be caused by a culmination of cardiac and neurologic factors, epilepsy induced bradycardia is a risk factor for SUDEP and may trigger lethal ventricular arrhythmias in susceptible myocardium. Development of a robust experimental model which recapitulates this would open the door for foundational studies to develop critical pharmacotherapies. SUDEP is a tragic outcome in patients with alternating hemiplegia of childhood (AHC) which is characterized by epilepsy, dystonia, paralysis, and, notably, sudden death in the setting of bradycardia. Among AHC patients, 90% harbor underlying pathologic genetic variants in the ATP1A3-encoded alpha-3 catalytic subunit of the Na/K ATPase pump (ATP1A3). We have identified a strong correlation between the most common AHC-associated ATP1A3 variant (D801N) and short QT on ECG and ventricular fibrillation during bradycardia. Human induced pluripotent stem cell-derived cardiac myocytes from an ATP1A3-D801N-positive child (hiPSC-CMD801N) demonstrate shortened repolarization time, disrupted calcium homeostasis, and delayed-after depolarizations, which are triggers for arrhythmias. Knock-in mice hosting the D801N variant (Atp1a3D801N) have seizures, bradycardia, and sudden death. Further, Atp1a3D801N mice have a predisposition to ventricular arrhythmias, particularly at lower heart rates, compared to controls. Collectively, these findings raise the possibility that disruption of ATP1A3 in the heart underlies SUDEP in AHC patients through bradycardia-triggered arrhythmias. We hypothesize that D801N reduces pump function leading to shortened repolarization time and predisposes to lethal ventricular arrhythmias. Our goal is to use AHC as a model to establish the role ATP1A3 in the heart, determine the mechanism of SUDEP, and explore the proarrhythmic effect of bradycardia. To approach this in an innovative and rigorous way, we will utilize patient-derived hiPSCD801N- and murine Atp1a3D801N-based models for in vitro, ex vivo, and in vivo studies to determine the mechanism of arrhythmia predisposition in the heart. Specifically, we propose to determine 1) the mechanism of action potential duration shortening induced by D801N in cardiac myocytes, 2) the mechanism of cardiac arrhythmogenesis induced by D801N in 3D tissue models and ex vivo analysis, and 3) whether bradycardia due to seizures is an arrhythmogenic trigger for a “vulnerable” myocardium in Atp1a3D801N mice. In accomplishing these aims and overall goal, we will determine the function of ATP1A3 in cardiovascular physiology and its role in cardiac repolarization, calcium signaling, and ventricular arrhythmias, thus identifying molecular targets for pharmacotherapy. Finally, this will develop robust and rigorous models to determine the mechanisms of sudden death in AHC and will provide insights into mechanisms of SUDEP more broadly.