Mechanisms of cardiac Ca dysregulation and arrhythmias in ankyrin B deficiency

锚蛋白 B 缺乏导致心脏 Ca 失调和心律失常的机制

• 批准号: 8685314
• 负责人: Sanda Despa
• 金额: $ 36.82万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8685314
• 关键词: ATP phosphohydrolase; Action Potentials; Adaptor Signaling Protein; Affect; Ankyrins; Arrhythmia; Bradycardia; Caffeine; Calpain; Cardiac; Cardiac Myocytes; Cations; Cells; Characteristics; Cleaved cell; Complex; Cytoskeleton; Dependence; Development; Frequencies; Functional disorder; Genetic; Goals; Heart; Human; Infarction; Inherited; Ion Channel; Ischemia; Lead; Long QT Syndrome; Measurement; Measures; Mediating; Membrane; Membrane Proteins; Messenger RNA; Molecular Biology Techniques; Mus; Muscle Cells; Myocardial Infarction; Na(+)-K(+)-Exchanging ATPase; Patients; Phenotype; Physiological; Protein Isoforms; Proteins; Proteolysis; Regulation; Reperfusion Therapy; Role; Sarcolemma; Sarcoplasmic Reticulum; Severities; Stress; Testing; Time; Ventricular; Ventricular Arrhythmia; Western Blotting; Wild Type Mouse; Work; cell injury; density; immunocytochemistry; loss of function; loss of function mutation; mutant; novel; null mutation; patch clamp; protein distribution; protein expression; sensor; sudden cardiac death; voltage

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.

描述（由申请方提供）：锚蛋白B（Ank B）是一种“衔接”蛋白，将几种膜蛋白锚定到细胞骨架上。几年前，当发现AnkB功能丧失突变产生人类4型长QT综合征（LQT 4）时，AnkB缺陷作为重要的促凝血因子出现，LQT 4是唯一由蛋白质而不是离子通道的改变产生的LQT。除了LQT 4之外，具有AnkB功能丧失突变的人显示复杂的心脏表型，其还包括心动过缓、应激诱导的室性心律失常和心脏性猝死。这种表型的机制在很大程度上是未知的。心肌梗死（MI）后梗死边缘区AnkB蛋白的表达和分布发生显著改变。这可能导致心肌梗死后重塑和心肌梗死发生的机制与AnkB功能丧失突变患者相似。与AnkB的直接相互作用是Na/Ca交换器（NCX）和Na/K-ATP酶（NKA）的膜靶向和稳定性所必需的，这在调节心脏[Na]i和[Ca]i以及因此的收缩性和触发心律失常的可能性中是必不可少的。由各种AnkB功能丧失突变体产生的人类心脏表型的严重性直接涉及这些突变体不能将NCX和NKA正确靶向心肌细胞肌膜。因此，改变的NCX和NKA表达和膜分布是由AnkB功能丧失产生的心脏表型的关键。这种表型在AnkB无效突变杂合小鼠（AnkB小鼠）中大量复制。来自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负荷以及早期后除极（EAD）和延迟后除极（DAD）频率增加。尽管其生理和病理生理意义，AnkB在调节心脏[Ca]i和心肌发生中的作用知之甚少。该提案的总体目标是破译负责改变心脏Ca调节和触发AnkB功能丧失（遗传和获得性）诱导的心律失常的机制。我们将结合联合收割机测量[Na]i和[Ca]i（在本体和连接裂隙），膜片钳和分子生物学技术在三个特定的目的在分离的心肌细胞。首先，我将测试几个具体的假设，旨在了解AnkB如何影响心脏细胞内的钙。目的2：探讨AnkB缺乏的心肌细胞发生早期和延迟后除极的机制。在最后的目标，我将测试的假设，即AnkB蛋白水解钙蛋白酶和随之而来的重塑NCX和NKA是一个更普遍的机制触发室性心律失常。这些研究将推进我们对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，这些信息可能导致开发与遗传性或获得性AnkB功能丧失相关的室性心律失常患者的新治疗方法。