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: 8787535
• 负责人: Sanda Despa
• 金额: $ 33.96万
• 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/8787535
• 关键词: Mechanisms; cardiac; Ca; dysregulation; arrhythmias

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(申请人提供)：Ankyrin B(AnkB)是一种将几种膜蛋白锚定在细胞骨架上的“接头”蛋白质。几年前，当发现AnkB功能丧失突变导致人类长QT综合征4型(LQT4)时，AnkB缺乏成为一个重要的促心律失常因素，LQT4是唯一由离子通道以外的蛋白质变化产生的LQT。除了LQT4，AnkB功能丧失突变的人类还表现出复杂的心脏表型，还包括心动过缓、应激性室性心律失常和心脏性猝死。导致这种表型的机制在很大程度上是未知的。心肌梗死(MI)后，心肌梗死边缘区AnkB蛋白的表达和分布发生了显著变化。这可能会导致心肌梗死后重塑和心律失常的发生机制，类似于AnkB功能丧失突变患者的机制。Na/Ca交换器(NCX)和Na/K-ATPase(NKA)的膜靶向性和稳定性需要与AnkB的直接相互作用，而NKA和NKA在调节心脏[Na]i和[Ca]i，从而调节收缩能力和触发心律失常的可能性中是必不可少的。各种AnkB功能缺失突变体产生的人类心脏表型的严重程度直接与这些突变体不能正确靶向心肌细胞肌膜NCX和NKA有关。因此，NCX和NKA的表达和膜分布的改变是AnkB功能丧失所产生的心脏表型的关键。这种表型主要在AnkB基因零突变杂合的小鼠(AnkB小鼠)中复制。AnkB鼠心肌细胞NCX和NKA蛋白表达略有降低，主要位于T管，细胞和肌浆网(SR)钙负荷较大，早期后除极(EAD)和延迟后除极(DAD)频率增加。尽管AnkB具有生理和病理生理学意义，但对其在心脏[Ca]i调节和心律失常发生中的作用却知之甚少。这项建议的总体目标是破译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。我们将在三个特定的目标中结合分离的心肌细胞的[Na]i和[Ca]i的测量(在块状和连接性间隙中)、膜片钳和分子生物学技术。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将侧重于AnkB缺乏的心肌细胞发生早期和延迟后除极的机制。在最终目的中，我将检验这样一种假设，即Calain对AnkB蛋白的分解以及随后NCX和NKA中的重构是触发室性心律失常的更一般机制。这些研究都将促进我们对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，可能导致开发新的治疗与遗传性或获得性AnkB功能丧失相关的室性心律失常的方法。