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

描述(申请人提供)：530万美国人被诊断为心力衰竭，伴随着钙离子处理的改变和导致病理性心脏重塑的转录变化。其中，钙调节的钙调神经磷酸酶/NFAT通路是心肌肥厚发生发展的重要调节因子。心肌钙通道CaVI 2是兴奋-收缩偶联所必需的，它也可以定位于空泡信号微域，允许局部钙离子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CaV1.2在心脏基因调控中的作用，在目标1中，我们建议创建腺病毒和表达融合到小窝蛋白结合基序的RGK嵌合蛋白的转基因动物。RGK蛋白是一种小的GTP酶，当过度表达时，它通过直接与通道复合体结合来完全抑制高电压门控钙通道(如CaV1.2)。嵌合的RGK蛋白定位于小窝应该只允许对小窝2的靶向抑制，使我们能够使用报告程序观察钙离子调节的转录因子活性的变化，并通过基因芯片实验观察基因表达的整体变化。进一步的实验将确定抑制腔内CaV1.2是否对动物压力超负荷模型中的心力衰竭具有保护作用。最近在神经元中证明了CaVI-2裂解的C-末端可以作为一个受钙离子调节的转录因子发挥作用，但该片段的转录活性在心脏中尚未被研究。我们建议建立表达CCAT的腺病毒和转基因动物模型，通过基因芯片分析来确定CCAT在心脏转录调控中的作用，并通过检测CCAT在心力衰竭的压力超负荷模型中是保护性的还是不适应性的来进一步研究CCAT在疾病中的作用。这里提出的实验将大大增加我们对钙离子信号如何启动在心力衰竭过程中重要的转录变化的理解。外行语言：钙离子信号对于心脏病背后基因表达的变化很重要。为了研究心脏L钙通道在转录中的作用，我们将构建一个在信号微域抑制该通道的小鼠模型，并确定该通道群体对基因转录和心力衰竭的贡献。我们类似地研究了由CaVI2.2C末端编码的转录因子在心力衰竭小鼠模型中的过度表达在疾病中的作用。这项工作将有助于揭示心脏L型钙通道在疾病相关基因表达调控中的作用，并可能为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.