CaMKII Dysregulation and Arrhythmias in Heart Failure

描述（由申请人提供）：心力衰竭（HF）中Ca 2 +/钙调蛋白（CaM）依赖性激酶II（CaMKII）的激活有助于心肌细胞凋亡、早期去极化后诱导的心律失常和适应不良重塑。在心脏中，大多数激活的CaMKII与肌膜相关，我们已经证明CaMKII与CaV1.2（L型）Ca 2+通道的孔形成11 C亚基结合，在那里它作为Ca 2+信号的专用整合剂发挥作用。CaMKII也是β 2-肾上腺素能刺激的CaV1.2 Ca 2+通道依赖性和PKA非依赖性下游效应物，因此将CaMKII置于HF中受影响的两个主要信号传导途径的会聚处：肾上腺素能刺激和Ca 2+失调。基于最近的结构信息和我们的新的生化数据，我们提出了一种新的模型，CaMKII和11 C之间的相互作用，提供了深入了解其功能和调节，并提供了一个基础，了解如何CaMKII的活动可以在HF扰动。本提案的目的是确定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。我们提出三个具体目标。在目的1中，我们将测试的假设，CaMKII的调节，并与CaV1.2钙离子通道的相互作用，涉及模仿的CaMKII自身抑制肽（AIP）。各种复杂的生物化学和生物物理技术将被用来测试这种新的模拟模型，在该模型中，我们提出，CaMKII与伪AIP域在11 C N和/或C末端的相互作用，钙调素或钙调素样钙结合蛋白结合到这些11 C域作为钙离子传感器，调节CaMKII和微调的调节。基于这些发现，在目标2中，我们将测试CaMK II位于HF中扰动的两个主要信号传导途径的会聚点的假设，2-肾上腺素能激活和细胞内Ca 2+信号传导，其性质是与心肌细胞中CaV1.2 Ca 2+的束缚相互作用。使用新的策略，以产生功能性的“敲入”的CaV1.2亚基在培养的心肌细胞，我们将测试的后果扰乱四级结构的CaMKII，CaV1.2，CaM和/或其他钙离子结合蛋白的CaMKII和CaV1.2的双向调节和PKA依赖性的2-肾上腺素调节的CaMKII信号。然后，这些数据将使我们能够应用我们的模型在目标3中测试HF干扰CaMKII/CaV1.2栓系相互作用中固有的双向调节的假设。我们将采用几种HF小鼠模型，这些模型是通过β 2-肾上腺素能信号传导途径的高“通量”引起的。我们假设HF改变了CaMKII和CaV1.2 Ca 2+通道之间的束缚，导致CaMKII活性的错误定位和失调，因此促进了与HF相关的病理生理后果。这项工作不仅将导致更好地了解心脏生理学，但它将提供新的见解心力衰竭和心脏功能受损。公共卫生相关性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和导致心源性猝死风险增加的后果尚不清楚。最近，在心力衰竭中，Ca 2 +/钙调蛋白（CaM）依赖性激酶II（CaMKII）的激活已被证明有助于心肌细胞凋亡，早期去极化后诱导的心律失常和适应不良重塑，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活性是由CaMKII与L-型（CaV1.2）Ca 2+通道的连接引起的。在这里，我们测试了一种新的模型CaMKII拴系CaV1.2通道，并提出HF改变这种动态，导致异常CaMKII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.