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.
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.
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