AKAP Regulation of PKA Targeting in the Heart

描述（由申请人提供）：我们正在根据通知号no - od -09-058提交R01 AG 16613的一年竞争性修订，通知标题：“NIH宣布竞争性修订申请的恢复法案资金可用性。”这一竞争性修订支持母体R01 Ag16613范围的显著扩展。亲本R01研究了a激酶锚定蛋白（AKAP）在离体大鼠心肌细胞和正常和衰竭大鼠心脏中对camp依赖性蛋白激酶（PKA）的调控和靶向作用，并评估了PKA靶向作用中断的功能意义。在这个新的特异性目标4中，我们将研究在心脏中，新的核AKAP，染色体结构域解旋酶结合蛋白8 (Chd8)，在缺血性损伤后心脏重塑过程中调节基因转录的作用。Chd8与介导细胞周期进程、凋亡和存活的多种因子相互作用，我们通过噬菌体展示、PKA调控亚基RII的共免疫沉淀鉴定出Chd8是一个AKAP；以及通过将RII结合位点突变为非活性脯氨酸衍生物而破坏RII的结合；我们通过定量PCR （qPCR）发现Chd8 mRNA和蛋白在心脏发育过程中表达，Chd8存在于心肌细胞的细胞核中；我们提供的证据表明，Chd8的表达在衰竭的心脏中比在正常的心脏中更大。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，我们预测心脏中PKA与Chd8的结合在Chd8依赖的基因转录调节中起着关键的调节作用。我们将验证以下假设：在大鼠心肌梗死后，Chd8在心脏重塑过程中上调并导致基因表达变化；我们还预测Chd8的激活受RII的结合调节。这些实验扩展了母体R01的原始目标，但并不与之重叠。本竞争性修订中所建议的亲本补助金活动包括：(i)定量PCR；（ii）诱导大鼠心肌缺血（MI）；（iii）在体内将腺病毒基因转入正常和衰竭大鼠心脏；（iv）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）腺病毒基因转移后大鼠心脏低温切片显微镜观察。在（新）特异性Aim 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)在心肌梗死诱导后，与对照组和对照组相比，鉴定体内大鼠心脏中上游调控序列与Chd8结合并受其调控的基因；（ii）将这些基因分成亚类：心力衰竭；细胞周期与增殖；细胞凋亡;（iii）在对照组、假手术大鼠和心肌梗死大鼠中研究这些家族中基因的差异转录；在Specific Aim 4.2中，我们将(i) ha标记的Chd8或（ii） Chd8的脯氨酸衍生物Chd8- p（取消RII结合）进行腺病毒（Ad）基因转移到对照、假性和心肌梗死大鼠心脏中。超声检查梗死范围及心功能；（iii） Chd8， PKA和将定位于正常和缺血大鼠左心室（LV）的低温切片。

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.