CaMKII Dysregulation and Arrhythmias in Heart Failure

描述（由申请人提供）：心力衰竭（HF）中Ca2+/钙调素（CaM）依赖性激酶II （CaMKII）的激活有助于心肌细胞凋亡，早期去极化诱导的心律失常和适应性重构不良。在心脏中，大多数活化的CaMKII与肌膜相关，我们已经证明CaMKII与CaV1.2 （l型）Ca2+通道的11C亚基结合，在那里它作为Ca2+信号的专用积分器起作用。CaMKII也是2-肾上腺素能刺激的CaV1.2 Ca2+通道依赖和pka独立的下游效应物，因此将CaMKII置于HF影响的两个主要信号通路的交汇处：高肾上腺素能刺激和Ca2+失调。基于最近的结构信息和我们新的生化数据，我们提出了CaMKII和11C之间相互作用的新模型，该模型提供了对其功能和调控的见解，并为理解CaMKII活性如何在HF中受到干扰提供了基础。本提案的目的是鉴定和测试这种相互作用的分子细节，并确定这种调节在HF中是如何改变的。我们提出三个具体目标。在Aim 1中，我们将验证CaMKII调节CaV1.2 Ca2+通道并与之相互作用涉及CaMKII自身抑制肽（AIP）的模仿的假设。各种复杂的生化和生物物理技术将被用来测试这种新的模拟模型，其中我们提出CaMKII与11C N和/或C末端的伪aip结构域相互作用，并且与这些11C结构域结合的cam或cam样Ca2+结合蛋白作为Ca2+传感器调节CaMKII并微调调节。在这些发现的基础上，在Aim 2中，我们将测试CaMKII位于HF中受2-肾上腺素能激活和细胞内Ca2+信号通路干扰的两个主要信号通路的交汇处的假设，这是由于其与心肌细胞中CaV1.2 Ca2+的栓系相互作用的性质。使用新的策略在培养的肌细胞中产生CaV1.2亚基的功能性“敲入”，我们将测试扰乱CaMKII、CaV1.2、CaM和/或其他Ca2+结合蛋白的四级结构对CaMKII和CaV1.2的双向调节以及对pka依赖性的CaMKII信号传导的2-肾上腺素能调节的影响。然后，这些数据将允许我们应用Aim 3中的模型来验证HF干扰CaMKII/CaV1.2系留相互作用中固有的双向调节的假设。我们将采用几种小鼠HF模型，这些模型通过2-肾上腺素能信号通路产生高“吞吐量”。我们假设HF改变了CaMKII和CaV1.2 Ca2+通道之间的束缚，导致CaMKII活性错位和失调，从而促进了与HF相关的病理生理后果。这项工作不仅将有助于更好地理解心脏生理学，而且将为心衰期间的心律失常和心功能受损提供新的见解。与公共卫生相关的声明心力衰竭每年影响约50万美国人，是发病率和死亡率的主要原因。心衰的分子机制和导致心源性猝死风险增加的后果尚不清楚。最近，Ca2+/钙调素（CaM）依赖性激酶II （CaMKII）在心力衰竭中的激活已被证明有助于心肌细胞凋亡，早期去极化诱导的心律失常和适应性重构，至少部分是由于2-肾上腺素能信号系统的意外刺激。我们最近的研究表明，正常的CaMKII活性是由于CaMKII与l型（CaV1.2） Ca2+通道的拴系造成的。在这里，我们测试了CaMKII与CaV1.2通道结合的新模型，并提出HF改变这种动态导致CaMKII异常激活。

DESCRIPTION (provided by applicant): Activation of Ca2+/calmodulin (CaM) dependent kinase II (CaMKII) in heart failure (HF) contributes to cardiomyocyte apoptosis, early-after depolarization-induced arrhythmias, and maladaptive remodeling. In heart, most activated CaMKII is associated with the sarcolemma and we have shown that CaMKII is bound to the pore-forming 11C subunit of CaV1.2 (L-type) Ca2+ channels, where it functions as a dedicated integrator of Ca2+ signals. CaMKII also is a CaV1.2 Ca2+ channel-dependent and PKA-independent downstream effector of 2-adrenergic stimulation, thus placing CaMKII at the convergence of the two major signaling pathways affected in HF: hyperadrenergic stimulation and Ca2+ dysregulation. Based upon recent structural information and our new biochemical data, we propose a novel model for the interaction between CaMKII and 11C that offers insight into its function and regulation and provides a basis for understanding how CaMKII activity could be perturbed in HF. The objective of this proposal is to identify and test the molecular details underlying this interaction, and determine how this regulation is altered in HF. We propose three specific aims. In Aim 1, we will test the hypothesis that CaMKII regulation of, and interaction with, CaV1.2 Ca2+ channels involves mimicry of the CaMKII autoinhibitory peptide (AIP). A variety of complex biochemical and biophysical techniques will be employed to test this novel mimicry model in which we propose that CaMKII interacts with pseudo-AIP domains in the 11C N and/or C termini and that, CaMs or CaM-like Ca2+-binding proteins bound to these 11C domains serve as the Ca2+-sensors that regulate CaMKII and fine-tune the regulation. Building upon those findings, in Aim 2 we will test the hypothesis that CaMKII sits at the convergence of two major signaling pathways perturbed in HF, 2-adrenergic activation and intracellular Ca2+ signaling, by nature of its tethered interaction with CaV1.2 Ca2+ in cardiomyocytes. Using novel strategies to generate functional "knock-ins" of CaV1.2 subunits in cultured myocytes, we will test the consequences of perturbing the quaternary structure of CaMKII, CaV1.2, CaM and/or other Ca2+-binding proteins upon the bidirectional regulation of CaMKII and CaV1.2 and upon the PKA-dependent 2-adrenergic regulation of CaMKII signaling. These data will then allow us to apply our model in Aim 3 to test the hypothesis that HF perturbs the bidirectional regulation inherent in the CaMKII/CaV1.2 tethered interaction. We will employ several mouse models of HF which result from high "throughput" through 2-adrenergic signaling pathways. We hypothesize that HF alters the tethering between CaMKII and CaV1.2 Ca2+ channels resulting in mislocalized and dysregulated CaMKII activity, therefore promoting the pathophysiological consequences associated with HF. Not only will this work will lead to a better understanding of cardiac physiology, but it will provide new insights into arrhythmogenesis and impaired cardiac function during HF. PUBLIC HEALTH RELEVANCE Statement Heart failure affects about 500, 000 Americans annually and is a leading cause of morbidity and mortality. The molecular mechanisms underlying heart failure and the consequences that lead to an increased risk of sudden cardiac death are not well understood. Recently, activation of Ca2+/calmodulin (CaM) dependent kinase II (CaMKII) in heart failure has been shown to contribute to cardiomyocyte apoptosis, early-after depolarization-induced arrhythmias, and maladaptive remodeling, at least in part to an unexpected stimulation by the 2-adrenergic signaling system. We have recently shown that normal CaMKII activity results from tethering of CaMKII to the L-type (CaV1.2) Ca2+ channel. Here we test a novel model for CaMKII tethering to CaV1.2 channels and propose that HF alters this dynamic to lead to abnormal CaMKII activation.