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AKAP Regulation of PKA Targeting in the Heart

AKAP 对心脏 PKA 靶向的调节

基本信息

批准号：
7814728
负责人：
Meredith Bond
金额：
$ 27.67万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7814728
关键词：
A kinase anchoring protein Adrenergic Agents Affect Affinity Apoptosis Binding Binding Proteins Binding Sites Cardiac Cardiac Myocytes Cardiac Myosins Cell Cycle Cell Cycle Progression Cell Nucleus Chromatin Co-Immunoprecipitations Complex Cyclic AMP Cyclic AMP-Dependent Protein Kinases Detection Development Dilated Cardiomyopathy Disease Echocardiography Family Functional disorder Funding Gene Expression Gene Transfer Genes Genetic Transcription Goals Heart Heart failure High Prevalence Human Immunofluorescence Immunologic Injury Label Left ventricular structure M-Mode Echocardiography Mediating Messenger RNA Microscopy Molecular Mutation Myocardial Infarction Myocardial Ischemia Nuclear Parents Pathology Patients Pattern Phage Display Phosphorylation Play Process Protein Kinase Proteins Publishing Rattus Recovery Regulation Role Signal Transduction TP53 gene Testing Troponin I United States National Institutes of Health adrenergic aging population cell growth chromatin immunoprecipitation chromatin protein chromatin remodeling cryostat cyclin E2 helicase in vivo mortality myosin-binding protein C novel parent grant promoter protein kinase A kinase public health relevance research study response

项目摘要

DESCRIPTION (provided by applicant): We are submitting a One Year Competing Revision of R01 AG 16613 according to Notice Number NOT-OD-09-058, Notice Title: "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. This competing revision supports a significant expansion of the scope of the parent R01 Ag16613. The parent R01 investigates regulation and targeting of cAMP-dependent protein kinase (PKA) by A-kinase anchoring proteins (AKAP) in isolated rat cardiac myocytes and in normal and failing rat heart and assesses the functional implications of disruption of PKA targeting. In this new Specific Aim 4, we will investigate the role, in the heart, of the novel nuclear AKAP, chromodomain helicase binding protein 8 (Chd8), in regulating gene transcription during cardiac remodeling following ischemic injury. Chd8 interacts with several factors that mediate cell cycle progression, apoptosis and survival, We identified Chd8 as an AKAP by phage display, by co- immunoprecipitation by the regulatory subunit RII of PKA; and disruption of RII binding by mutation of the RII binding site to an inactive prolinated derivative; we showed by quantitative PCR (qPCR) that Chd8 mRNA and protein are expressed in the heart during development and Chd8 is found in the nucleus of cardiac myocytes; we provide evidence that Chd8 expression is greater in failing than non-failing human hearts.. By analogy with nuclear AKAP95, where chromatin binding depends in turn on binding of phosphorylated RII (at T54) to AKAP95, we predict that PKA binding to Chd8 in the heart plays a critical regulatory role in Chd8 dependent-regulation of gene transcription. We will test the hypothesis that following an MI in rats, Chd8 is upregulated and contribute to changes in gene expression, during cardiac remodeling; we also predict that Chd8 activation is regulated by binding of RII. These experiments extend but do not overlap the original aims of the parent R01. Activities in the parent grant that encompass those proposed in this Competing Revision include (i) quantitative PCR; (ii) induction of myocardial ischemia (MI) in rats; (iii) adenoviral gene transfer into normal and failing rat hearts in vivo; (iv) cardiac function assessment by transthoracic and M-mode echocardiography (echo) (v) immunofluorescence labeling and (vi) microscopy of cryostat sections of rat heart after adenoviral gene transfer. In (new) Specific Aim 4.1, we will use Chromatin Immunoprecipitation (ChIP) followed by high throughput sequencing (ChIP-seq) to: (i) identify genes whose upstream regulatory sequence binds to and is regulated by Chd8 in rat hearts in vivo, following induction of MI, vs shams and controls; (ii) organize these genes into subclasses: heart failure; cell cycle and proliferation; apoptosis; (iii) investigate differential transcription of genes within these families in controls, sham-operated and MI rats; in Specific Aim 4.2: we will perform adenoviral (Ad) gene transfer of (i) HA-tagged Chd8 or (ii) the prolinated derivative of Chd8, Chd8-P (with abrogated RII binding) into control, sham and MI rat hearts. Extent of infarct and cardiac function will be determined by echo; (iii) Chd8, PKA and will be localized in cryostat sections of left ventricle (LV) of normal and ischemic rat hearts. PUBLIC HEALTH RELEVANCE: The continued high prevalence of heart failure and resultant mortality, in the aging population demands the continuing and urgent need for more effective heart failure therapies. Therefore a more thorough understanding of the molecular processes underlying development of cardiac pathology will provide much-needed opportunities for detection and treatment of the disease. A primary change that underlies cardiac remodeling that occurs during heart failure is impaired activation of the cAMP/PKA signaling cascade. Our project investigates a novel nuclear AKAP protein, Chd8 that may participate in regulation of cardiac remodeling during ischemic heart failure.

描述（由申请人提供）：我们根据通知编号NOT-OD-09-058提交R 01 AG 16613的一年竞争修订版，通知标题：“NIH宣布恢复法案资金可用于竞争修订申请。该竞争修订版支持父R 01 Ag 16613范围的显著扩展。母体R 01研究了A-激酶锚定蛋白（AKAP）在分离的大鼠心肌细胞以及正常和衰竭大鼠心脏中对cAMP依赖性蛋白激酶（PKA）的调节和靶向作用，并评估了PKA靶向作用中断的功能意义。在这个新的具体目标4，我们将调查的作用，在心脏中，新的核AKAP，chromodomain解旋酶结合蛋白8（Chd 8），在调节基因转录在心脏重塑缺血性损伤后。Chd 8与介导细胞周期进程、凋亡和存活的几种因子相互作用。我们通过噬菌体展示、PKA的调节亚基RII的免疫共沉淀以及通过RII结合位点突变为无活性脯氨酸化衍生物来破坏RII结合，鉴定了Chd 8为AKAP。定量PCR（qPCR）结果表明，Chd 8 mRNA和蛋白质在发育过程中的心脏中均有表达，Chd 8定位于心肌细胞核;我们提供的证据表明，Chd 8的表达是更大的失败比非失败的人类心脏。通过与核AKAP 95类比，其中染色质结合依次依赖于磷酸化RII（在T54处）与AKAP 95的结合，我们预测PKA与心脏中Chd 8的结合在Chd 8依赖的基因转录调节中起关键的调节作用。我们将测试的假设，即在大鼠心肌梗死后，Chd 8上调，并有助于基因表达的变化，在心脏重塑;我们还预测，Chd 8的激活调节RII的结合。这些实验扩展了但不与母体R 01的原始目标重叠。包括本竞争修订版中提出的活动在内的母基金活动包括：（i）定量PCR;（ii）诱导大鼠心肌缺血（MI）;（iii）腺病毒基因转移到体内正常和衰竭大鼠心脏中;（iv）通过经胸和M型超声心动图（echo）进行心脏功能评估（v）免疫荧光标记和（vi）腺病毒基因转移后大鼠心脏的低温恒温切片的显微镜检查。在（新）Specific Aim 4.1中，我们将使用染色质免疫沉淀（ChIP），然后进行高通量测序（ChIP-seq）以：（i）在诱导MI后，与假手术和对照相比，在体内鉴定其上游调控序列结合至大鼠心脏中的Chd 8并受Chd 8调控的基因;（ii）将这些基因组织成亚类：心力衰竭;细胞周期和增殖;细胞凋亡;（iii）研究对照、假手术和MI大鼠中这些家族内基因的差异转录;在具体目标4.2中：我们将（i）HA标记的Chd 8或（ii）Chd 8的脯氨酸化衍生物Chd 8-P（具有废除的RII结合）的腺病毒（Ad）基因转移到对照、假手术和MI大鼠心脏中。梗塞的程度和心脏功能将通过超声心动图确定;（iii）Chd 8、PKA，并且将定位在正常和缺血大鼠心脏的左心室（LV）的低温恒温器切片中。公共卫生关系：老年人群中心力衰竭的持续高患病率和由此导致的死亡率要求持续且迫切需要更有效的心力衰竭疗法。因此，对心脏病理学发展的分子过程的更透彻的理解将为疾病的检测和治疗提供急需的机会。在心力衰竭期间发生的心脏重塑的基础的主要变化是cAMP/PKA信号级联的活化受损。我们的项目研究了一种新的核AKAP蛋白，Chd 8，可能参与缺血性心力衰竭期间心脏重塑的调节。