Targeting pathologic intracellular calcium release to prevent lethal arrhythmias

项目摘要/摘要 RyR2是一种细胞内钙释放通道，表达于肌浆网(SR)。 心肌细胞。在正常心脏，RyR2钙从SR释放受到严格调控，仅发生在 收缩，以促进心脏收缩。在心脏病中，RyR2钙释放可发生在舒张期和 被认为是病态的。病理性钙释放可由RyR2突变或RyR2翻译后引起 修改。由于肌浆网钙离子耗竭，舒张期病理性钙释放降低心肌收缩能力 由于钠(Na)流进入心肌细胞，导致延迟的后除极，因此储存并诱发心律失常 细胞通过钠钙交换器。RyR2基因突变致儿茶酚胺能多形性室性心动过速 (CPVT)，一种遗传性心律失常综合征，而翻译后修饰已被广泛记录 在心肌梗塞引起的充血性心力衰竭(CHF)中。这两种情况都与一种 心源性猝死(SCD)的风险。我的导师发现一种古老的抗心律失常药物-氟卡胺- 防止病理性钙释放而不是生理性钙释放，在预防脑室出血方面非常有效 CPVT患者的心律失常。重要的是，他最近发现氟卡胺的疗效不依赖于钠 通道阻塞，而不是RyR2阻塞。不幸的是，由于其钠通道阻滞性，氟卡胺 增加CHF患者的死亡率，不能用于这一患者群体。要解决这些问题 患者患SCD的风险-目前可用药物尚未降低-我的目标是开发一种氟卡胺类似物， 维持RyR2阻滞剂，但不维持钠通道阻滞剂。在此过程中，我将研究其作用机制。 并验证其疗效依赖于跨膜电位变化的假设 高级我的研究背景和膜片钳电生理和钙成像在临床中的应用 我导师的实验室将使我能够测试我们的合作者在合成中产生的氟卡胺类似物 化学反应。为了探索氟卡胺电压依赖的RyR2阻滞剂的作用机制，我将 使用各种工具，包括遗传编码的电压指示器和电压敏感染料来捕获 在SR处发生的理论膜电位变化。这一目标的结果不仅将澄清 氟卡胺的作用机制也产生了一种新的治疗原理--电压依赖 阻断RyR2通道是开发未来RyR2抑制剂AS的关键特征 抗心律失常药物。

PROJECT SUMMARY/ABSTRACT RyR2 is an intracellular calcium (Ca) release channel expressed in the sarcoplasmic reticulum (SR) of cardiomyocytes. In the normal heart, RyR2 Ca release from the SR is tightly regulated and only occurs during systole to facilitate heart contraction. In heart disease, RyR2 Ca release can occur during diastole and is considered pathologic. Pathologic Ca release can be caused by RyR2 mutations or RyR2 post-translational modifications. Pathologic Ca release during diastole reduces cardiac contractility due to depletion of SR Ca stores and is pro-arrhythmogenic due to delayed after-depolarizations resulting from sodium (Na) flux into the cell via the Na-Ca exchanger. RyR2 mutations cause catecholaminergic polymorphic ventricular tachycardia (CPVT), a genetic arrhythmia syndrome, while post-translational modifications have been widely documented in congestive heart failure (CHF) caused by myocardial infarction. Both conditions are associated with a high risk of sudden cardiac death (SCD). My mentor discovered that an old antiarrhythmic drug – flecainide – prevents pathologic rather than physiologic Ca release and is strikingly effective in preventing ventricular arrhythmias in CPVT patients. Importantly, he recently discovered that flecainide’s efficacy depends not on Na channel block but rather RyR2 block. Unfortunately, due to its Na channel blocking properties, flecainide increases mortality in patients with CHF and cannot be used in this patient population. To address these patients’ risk for SCD – currently unmitigated by available drugs – I aim to develop a flecainide analogue that maintains RyR2 block but not Na channel block. In doing so, I will investigate the mechanism of action of flecainide and test the hypothesis that its efficacy depends on a change in the membrane potential across the SR. My research background and the established use of patch clamp electrophysiology and calcium imaging in my mentor’s lab will enable me to test flecainide analogues generated by our collaborators in synthetic chemistry. To probe the mechanism of action underlying flecainide’s voltage-dependent RyR2 block, I will employ a variety of tools including genetically encoded voltage indicators and voltage-sensitive dyes to capture the theoretical membrane potential change that occurs at the SR. The results from this aim will not only clarify flecainide’s mechanism of action but also yield a novel therapeutic principle – that voltage-dependent block of RyR2 channels is a key feature for the development of future RyR2 inhibitors as antiarrhythmic drugs.