AKAP Regulation of PKA Targeting in the Heart

描述(由申请人提供)：我们根据通知编号NOT-OD-09-058，提交R01 AG 16613的一年竞争性修订，通知标题：“美国国立卫生研究院宣布恢复法案资金可用于竞争性修订申请。这一竞争版本支持父R01 Ag16613范围的显著扩展。亲本R01研究了A-激酶锚定蛋白(AKAP)对分离的大鼠心肌细胞以及正常和衰竭大鼠心脏中cAMP依赖的蛋白激酶(PKA)的调节和靶向性，并评估了PKA靶向性中断的功能意义。在这个新的特定目标4中，我们将研究新的核AKAP，染色域解旋酶结合蛋白8(CHD8)在心脏缺血损伤后心脏重塑过程中调节基因转录的作用。CHD8与多种调节细胞周期进程、细胞凋亡和存活的因子相互作用，我们通过噬菌体展示、PKA调节亚基RII的免疫共沉淀鉴定CHD8为AKAP；通过RII结合位点突变破坏RII结合来破坏RII结合；我们通过定量聚合酶链式反应(QPCR)显示CHD8的mRNA和蛋白在发育过程中在心脏中表达，CHD8在心肌细胞核中发现；我们提供的证据表明，CHD8在衰竭的心脏中的表达高于未衰竭的心脏。通过与核AKAP95类似，其中染色质结合反过来依赖于磷酸化的RII(在T54)与AKAP95的结合，我们预测PKA与心脏中CHD8的结合在CHD8依赖的基因转录调控中发挥关键调节作用。我们将检验这一假设，即在大鼠心肌梗死后，CHD8上调并有助于心脏重塑期间基因表达的变化；我们还预测CHD8的激活受RII结合的调节。这些实验延伸了父R01的原始目标，但不与其重叠。母基金的活动包括：(I)定量聚合酶链式反应；(Ii)诱导大鼠心肌缺血；(Iii)在体将腺病毒基因转移到正常和衰竭的大鼠心脏；(Iv)通过经胸和M型超声心动图(ECHO)评估心功能；(V)免疫荧光标记和(Vi)腺病毒基因转移后的大鼠心脏冷冻切片的显微镜观察。在(新的)特定目标4.1中，我们将使用染色质免疫沉淀(CHIP)和高通量测序(CHIP-SEQ)来：(I)在MI、VS Sham和对照组诱导后，在活体大鼠心脏中鉴定其上游调控序列与CHD8结合并受CHD8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)在对照、假手术和MI大鼠中，研究这些家族中基因的差异转录；在特定的目标4.2中，我们将(I)HA标记的CHD8或(Ii)CHD8的脯氨酸衍生物CHD8-P(与RII结合被取消)进行腺病毒(Ad)基因转移到对照、假手术和心肌梗死大鼠心脏。心肌梗死范围和心功能将通过ECHO确定；(Iii)CHD8、PKA和将定位于正常和缺血大鼠心脏的左心室(LV)冷冻切片。 与公共卫生相关：在老龄化人口中，心力衰竭的患病率和由此导致的死亡率持续高企，这就要求对更有效的心力衰竭疗法的持续和迫切需求。因此，更透彻地了解心脏病理发展的分子过程将为发现和治疗这种疾病提供亟需的机会。心力衰竭时发生的心脏重构的一个主要变化是cAMP/PKA信号级联的激活受损。我们的项目研究了一种新的核AKAP蛋白CHD8，它可能参与缺血性心力衰竭时心脏重构的调节。

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.