CaMKII Dysregulation and Arrhythmias in Heart Failure

描述(由申请人提供)：在心力衰竭(HF)中，钙/钙调蛋白(CaM)依赖的激酶II(CaMKII)的激活有助于心肌细胞的凋亡、去极化早期-后诱发的心律失常和适应不良的重构。在心脏中，大多数激活的CaMKII与肌膜有关，我们已经证明CaMKII与CaV1.2(L类型)钙通道的11C亚基结合，在那里它作为钙信号的专用积分器发挥作用。CaMKII也是CaV1.2钙通道依赖性和PKA非依赖性的2-肾上腺素能刺激下游效应器，从而使CaMKII处于心力衰竭两条主要信号通路的汇合点：高肾上腺素能刺激和钙离子失调。基于最近的结构信息和我们新的生化数据，我们提出了一个新的模型来研究CaMKII与11C之间的相互作用，该模型提供了对其功能和调控的洞察，并为理解CaMKII活性如何在HF中受到干扰提供了基础。这项建议的目标是识别和测试这种相互作用背后的分子细节，并确定这种调节在HF中是如何改变的。我们提出了三个具体目标。在目标1中，我们将检验这样的假设，即CaMKII对CaV1.2钙通道的调节和与其相互作用涉及CaMKII自身抑制肽(AIP)的模仿。我们将使用各种复杂的生化和生物物理技术来测试这一新的模拟模型，在该模型中，CaMKII与11C N和/或C末端的伪AIP结构域相互作用，与这些11C结构域结合的CaM或CaM样钙结合蛋白作为钙感受器调节CaMKII并微调调节。在这些发现的基础上，在目标2中，我们将检验这一假设，即由于CaMKII与心肌细胞中CaV1.2 Ca~(2+)的拴系相互作用，CaMKII位于两条主要的信号通路的交汇点上，这两条信号通路被干扰在HF中，即2-肾上腺素能激活和细胞内钙信号。我们将利用新的策略在培养的心肌细胞中产生CaV1.2亚单位的功能性“敲入”，以测试扰乱CaMKII、CaV1.2、CaM和/或其他钙结合蛋白的四级结构对CaMKII和CaV1.2的双向调节以及对依赖PKA的2-肾上腺素能调节CaMKII信号的影响。然后，这些数据将使我们能够将我们的模型应用于目标3，以测试HF扰乱CaMKII/CaV1.2拴系相互作用中固有的双向调节的假设。我们将采用几种通过2-肾上腺素能信号通路的高“吞吐量”所致的心力衰竭小鼠模型。我们假设，HF改变了CaMKII和CaV1.2钙通道之间的连接，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了解心脏生理学，而且将为心衰期间心律失常的发生和心功能受损提供新的见解。公共健康相关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。心力衰竭的分子机制以及导致心源性猝死风险增加的后果尚不清楚。最近，钙/钙调蛋白依赖的激酶II(CaMKII)在心力衰竭中的激活被证明与心肌细胞凋亡、去极化后早期心律失常和适应性重构有关，至少部分是由于2-肾上腺素能信号系统的意外刺激。我们最近发现，正常的CaMKII活性是CaMKII与L型钙通道(CaV1.2)连接的结果。在这里，我们测试了一个新的CaMKII连接到CaV1.2通道的模型，并提出HF改变了这一动态，导致了CaMKII的异常激活。

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.