Regulation of Cardiac Gene Expression by the L-type Calcium Channel, CaV1.2.

描述（由申请人提供）：530万美国人被诊断为心力衰竭，伴有Ca2+处理改变和转录改变，导致病理性心脏重构。其中一种途径是ca2 -t调节的钙调神经磷酸酶/NFAT，它是心肌肥厚发生的重要调节因子。心脏Ca2+通道CaVI。2，这是兴奋-收缩耦合所必需的，也可以定位于腔泡信号微域，允许局部Ca2+通过这些通道内流参与信号事件和转录调节，可能是通过钙调神经磷酸酶/NFAT。为了研究洞穴CaV1.2对心脏基因调控的贡献，在目的1中，我们提出创建一种腺病毒和转基因动物，表达融合到洞穴蛋白结合基序的嵌合RGK蛋白。RGK蛋白是一种小的gtpase，当过表达时，通过与通道复合物直接关联，完全抑制高压门控Ca2+通道（如CaV1.2）。嵌合RGK蛋白在小泡上的定位应该允许靶向抑制小泡CaVI。2只，允许我们使用报告基因法观察Ca2+调节的转录因子活性的变化，并通过基因芯片法观察基因表达的全局变化。进一步的实验将确定在动物压力过载模型中，CaV1.2的抑制是否对心力衰竭有保护作用。最近有研究表明，在神经元中，cai的C端被切割。2可以作为Ca2+调节的转录因子，然而，这个片段的转录活性，称为CCAT，在心脏中尚未研究。我们建议创建表达CCAT的腺病毒和转基因动物模型，通过基因芯片检测来确定其在心脏转录调控中的作用，并通过检测CCAT在心力衰竭压力过载模型中是保护还是不适应来进一步研究CCAT在疾病中的作用。这里提出的实验将大大增加我们对Ca2+信号如何启动心力衰竭期间重要的转录变化的理解。外行语言：Ca2+信号对导致心脏病的基因表达变化很重要。为了研究心脏l型Ca2+通道在转录中的作用，我们将构建一个小鼠模型，在信号微域中抑制该通道，并确定该通道群体对基因转录和心力衰竭的贡献。我们同样研究了由cai编码的转录因子的作用在心力衰竭小鼠模型中，c -末端在疾病中的过度表达。这项工作将有助于揭示心脏l型Ca2+通道在调节与疾病相关的基因表达中的作用，并可能为对抗心力衰竭提供新的翻译策略。

DESCRIPTION (provided by applicant): 5.3 million Americans are diagnosed with heart failure, which is accompanied by altered Ca2+ handling and transcriptional changes leading to pathological cardiac remodeling. One such pathway, Ca2-t-regulated calcineurin/NFAT, is an essential regulator for the development of cardiac hypertrophy. The cardiac Ca2+ channel CaVI .2, which is required for excitation-contraction coupling, can also be localized to caveolar signaling microdomains that allow local Ca2+ influx through these channels to participate in signaling events and transcriptional regulation, perhaps via calcineurin/NFAT. To examine the contribution of caveolar CaV1.2 to cardiac gene regulation, in aim 1 we propose to create an adenovirus and transgenic animal expressing a chimeric RGK protein fused to a caveolin-binding motif. RGK proteins are small GTPases that completely inhibit high voltage-gated Ca2+ channels (such as CaV1.2) by direct association with the channel complex when overexpressed. Localization of the chimeric RGK protein to caveolae should allow targeted inhibition of caveolar CaVI .2 only, allowing us to observe changes in activity in Ca2+-regulated transcription factors using reporter assays, and global changes in gene expression by gene chip assay. Further experimentation will determine whether inhibition of caveolar CaV1.2 is protective against heart failure in an animal pressure overload model. Recently it was demonstrated in neurons that the cleaved C- terminus of CaVI .2 can function as a Ca2+-regulated transcription factor, however transcriptional activity of this fragment, termed CCAT, is unstudied in the heart. We propose creation of adenoviruses and transgenic animal models expressing CCAT to determine its contribution to transcriptional regulation in the heart by means of gene chip assay and further examination of CCAT roles in disease by examining whether it is protective or maladaptive in a pressure overload model of heart failure. Experiments proposed here will substantially increase our understanding of how Ca2+ signals initiate transcriptional changes important during heart failure. Lay language: Ca2+ signals are important for changes in gene expression that underlie heart disease. To examine the role of the cardiac L-type Ca2+ channel in transcription, we will construct a mouse model inhibiting this channel in signaling microdomains and determine the contribution of this channel population to gene transcription and heart failure. We similarly examine the role of the transcription factor encoded by the CaVI.2 C-terminus in disease by overexpression in a mouse model of heart failure. This work will help reveal the role of the cardiac L-type Ca2+ channel in regulation of gene expression related to disease and may suggest new translational strategies to combat heart failure.