Mechanisms of cardiac Ca dysregulation and arrhythmias in ankyrin B deficiency

描述（由申请人提供）：锚蛋白B （AnkB）是一种“适配器”蛋白，可将几种膜蛋白锚定在细胞骨架上。几年前，当发现AnkB功能缺失突变产生人类长QT综合征4型（LQT4）时，AnkB缺乏症成为一个重要的促心律失常因素，LQT4是唯一一种由离子通道以外的蛋白质改变产生的LQT。除了LQT4外，AnkB功能缺失突变的人还表现出复杂的心脏表型，包括心动过缓、应激性室性心律失常和心源性猝死。造成这种表型的机制在很大程度上是未知的。心肌梗死（MI）后，AnkB蛋白在梗死边界区表达和分布发生显著改变。这可能导致心肌梗死后重构和心律失常发生的机制与AnkB功能缺失突变患者相似。Na/Ca交换器（NCX）和Na/ k - atp酶（NKA）的膜靶向和稳定性需要与AnkB直接相互作用，而NKA是调节心脏[Na]i和[Ca]i以及收缩性和诱发心律失常的潜力所必需的。各种AnkB功能丧失突变体产生的人类心脏表型的严重程度与这些突变体无法将NCX和NKA正确靶向心肌细胞肌膜直接相关。因此，NCX和NKA表达和膜分布的改变是AnkB功能丧失引起的心脏表型的关键。这种表型在AnkB的零突变的杂合小鼠（AnkB小鼠）中大量复制。AnkB小鼠的肌细胞显示NCX和NKA蛋白表达适度降低，主要在t小管，较大的细胞和肌浆网（SR） Ca负荷和早期后去极化（EAD）和延迟后去极化（DAD）频率增加。尽管其具有生理和病理生理意义，但AnkB在调节心脏[Ca]i和心律失常中的作用尚不清楚。该建议的总体目标是破译由AnkB功能丧失（遗传性和获得性）引起的心脏钙调节改变和触发心律失常的机制。我们将结合[Na]i和[Ca]i的测量（在大块和结裂中），膜片钳和分子生物学技术在三个特定目的中分离心肌细胞。首先，我将测试几个特定的假设，旨在了解AnkB如何影响心脏细胞内钙。目的2将关注AnkB缺乏的肌细胞早期和延迟后去极化发生的机制。在最后的目的中，我将检验一个假设，即calpain对AnkB蛋白的水解以及NCX和NKA中随后的重塑是触发室性心律失常的更普遍的机制。这些研究将促进我们对AnkB如何影响心脏[Ca]i调节的理解，并将提供关键的机制信息，可能导致与遗传或获得性AnkB功能丧失相关的室性心律失常患者的新治疗方法的开发。

DESCRIPTION (provided by applicant): Ankyrin B (AnkB) is an "adaptor" protein that anchors several membrane proteins to the cytoskeleton. AnkB deficiency emerged as an important pro-arrhythmic factor a few years ago, when it was found that AnkB loss- of-function mutations generate human long QT syndrome type 4 (LQT4), the only LQT produced by alterations in a protein other than an ion channel. Besides LQT4, humans with AnkB loss-of-function mutations display a complex cardiac phenotype that also includes bradycardia, stress-induced ventricular arrhythmias and sudden cardiac death. The mechanisms responsible for such a phenotype are largely unknown. AnkB protein expression and distribution are drastically altered in the infarct border zone after myocardial infarction (MI). This may result in a mechanism for post-MI remodeling and arrhythmogenesis similar to that found in patients with AnkB loss-of-function mutations. Direct interaction with AnkB is required for the membrane targeting and stability of the Na/Ca exchanger (NCX) and Na/K-ATPase (NKA), which are essential in the regulation of cardiac [Na]i and [Ca]i and thus contractility and potential for triggered arrhythmias. The severity of the human cardiac phenotype generated by various AnkB loss-of-function mutants directly relates to the inability of those mutants to target NCX and NKA correctly to the cardiac myocyte sarcolemma. Thus, altered NCX and NKA expression and membrane distribution are key to the cardiac phenotype generated by AnkB loss-of-function. This phenotype is largely reproduced in mice heterozygous for a null mutation in AnkB (AnkB mice). Myocytes from AnkB mice show modestly reduced NCX and NKA protein expression, predominantly at the T-tubules, larger cellular and sarcoplasmic reticulum (SR) Ca load and increased frequency of early afterdepolarizations (EAD) and delayed afterdepolarizations (DAD). Despite its physiological and pathophysiological significance, the role of AnkB in regulating cardiac [Ca]i and arrhythmogenesis is poorly understood. The overall goal of this proposal is to decipher the mechanisms responsible for altered cardiac Ca regulation and triggered arrhythmias induced by AnkB loss-of-function (inherited and acquired). We will combine measurements of [Na]i and [Ca]i (in the bulk and junctional cleft), patch-clamp and molecular biology techniques in isolated cardiac myocytes in three Specific Aims. First, I will test several specific hypotheses aimed at understanding how AnkB affects intracellular Ca in the heart. Aim 2 will focus on the mechanisms responsible for the occurrence of early and delayed afterdepolarizations in myocytes with AnkB deficiency. In the final aim I will test the hypothesis that AnkB proteolysis by calpain and the ensuing remodeling in NCX and NKA is a more general mechanism for triggered ventricular arrhythmias. These studies will both advance our understanding of how AnkB affects cardiac [Ca]i regulation and will provide key mechanistic information that could lead to the development of new treatments for patients with ventricular arrhythmias associated with inherited or acquired AnkB loss-of-function.