ATF4 a Novel Regulator of Cardiac Development

ATF4 心脏发育的新型调节剂

• 批准号: 10657081
• 负责人: Ju Chen
• 金额: $ 55.3万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10657081
• 关键词: Ablation; Address; Adult; Appearance; Atrial Fibrillation; Binding; Binding Sites; Biological Process; Cardiac; Cardiac Myocytes; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cellular Stress; Cessation of life; Complex; Cyclic AMP-Responsive DNA-Binding Protein; DNA; Data; Defect; Development; Dilated Cardiomyopathy; Dimensions; Disease; Down-Regulation; Embryo; Exhibits; Family; Family member; Genes; Genetic Transcription; Heart; Heart Abnormalities; Heart Diseases; Heart failure; Knock-out; Knockout Mice; Knowledge; Left; Leucine Zippers; Maintenance; Malignant Neoplasms; Mediating; Mediator; Messenger RNA; Modeling; Molecular; Morphogenesis; Morphology; Mus; Neonatal; Pathologic; Pathway interactions; Patients; Perinatal mortality demographics; Pharmacologic Substance; Phenotype; Physiological; Physiology; Play; Proliferating; Protein Family; Proteins; Regulation; Regulator Genes; Reporting; Role; Series; TP53 gene; Tamoxifen; Therapeutic; Thick; Thinness; Tissues; Transgenic Mice; Troponin T; Up-Regulation; Ventricular; Xenopus laevis; activating transcription factor; activating transcription factor 4; biological adaptation to stress; cardiogenesis; cell type; chromatin immunoprecipitation; congenital heart disorder; genome-wide; heart function; in vivo; inhibitor; mouse model; myosin light chain 2; new therapeutic target; novel; pharmacologic; postnatal; programs; side effect; transcription factor; transcriptome sequencing

PROJECT SUMMARY Activating Transcription Factor 4 (ATF4), also known as cAMP-Response Element Binding Protein 2 (CREB2), belongs to the ATF/cAMP responsive element-binding (CREB) protein family of transcription factors. As a well- characterized stress-response transcription factor, ATF4 is constitutively and ubiquitously expressed at low levels but can be rapidly induced under a variety of cell-stress conditions. Previous studies have shown that ATF4 functions in diverse cell types and tissues under various physiological and pathological conditions, including cardiac diseases. However, little is known as to the specific role of ATF4 and its target genes in mammalian cardiac development. To address this gap in knowledge and to determine the role of ATF4 in cardiomyocytes (CMs), we have generated novel Atf4 CM-specific constitutive knockout (cKO) and tamoxifen- inducible CM-specific knockout (icKO) mouse lines. Our preliminary studies revealed that Atf4 cKO mice exhibited perinatal death and cardiac morphological defects, associated with reduced CM proliferation. RNA- seq and ChIP-seq analyses of embryonic ventricular tissues revealed upregulation of a series of cell cycle arrest-associated genes known to be downstream of p53, and downregulation of a series of p53-independent cardiac development/function and/or cell cycle progression associated genes, most of which harbored ATF4 binding regions, in Atf4 cKO mice. Loss of ATF4 in developing CMs also resulted in increased p53 protein levels but not Trp53 mRNA levels. Moreover, p53 ablation in Atf4 cKO mice partially restored ventricular wall thickness and ameliorated upregulation of p53 target cell cycle arrest genes at E17.5, but failed to rescue lethality beyond postnatal day 1. Conversely, inducible ablation of Atf4 in adult CMs had no effect on cardiac function or left ventricular dimension, suggesting distinct roles for ATF4 at specific stages of CM development. Taken together, the foregoing evidence leads us to the hypothesis that ATF4 plays an essential role in CM proliferation and function via p53-dependent and -independent mechanisms at specific stages of cardiac development. Accordingly, our Specific Aims are: 1. To determine the role of ATF4 in cardiomyocyte proliferation and cardiac development by analyzing Atf4 cardiomyocyte-specific knockout (Atf4 cKO) mice, and to elucidate mechanisms by which ATF4 regulates target gene pathways in a p53-dependent and/or -independent manner; and 2. To determine times at which ATF4 is required for embryonic and neonatal cardiomyocyte proliferation and function (from E7.5 to P30) by analysis of Atf4 inducible cardiomyocyte- specific knockout (icKO) mice. Our proposed studies will help us to understand the specific roles of ATF4 and p53, as well as other novel ATF4 targets in CMs at critical developing stages in vivo, as well as to determine a safe therapeutic window for the potential application of ATF4 and/or p53 inhibitors in cardiac diseases.

项目摘要 激活转录因子4（ATF4），也称为CAMP响应元件结合蛋白2（CREB2）， 属于转录因子的ATF/CAMP响应元素结合（CREB）蛋白家族。作为一个很好的 表征应力反应转录因子，ATF4在低点且普遍于低表达 水平但可以在各种细胞压力条件下迅速诱导。先前的研究表明 在各种生理和病理条件下，ATF4在各种细胞类型和组织中起作用， 包括心脏疾病。但是，几乎不知道ATF4及其靶基因在 哺乳动物心脏发展。解决这一知识的差距，并确定ATF4在 心肌细胞（CMS），我们已经产生了新型的ATF4 CM特异性敲除（CKO）和他莫昔芬 - 诱导CM特异性敲除（ICKO）小鼠系。我们的初步研究表明ATF4 CKO小鼠 与CM增殖减少有关，表现出围产期死亡和心脏形态缺陷。 RNA- 胚胎心室组织的SEQ和CHIP-SEQ分析显示了一系列细胞周期的上调 已知的与逮捕相关的基因是p53的下游，并下调了一系列p53独立的 心脏发育/功能和/或细胞周期进展相关基因，其中大多数藏有ATF4 结合区域，在ATF4 CKO小鼠中。发育中的CMS中ATF4的损失也导致p53蛋白增加 水平，但不是TRP53 mRNA水平。此外，ATF4 CKO小鼠中的p53消融部分恢复了心室壁 p53目标细胞周期停滞基因在E17.5处的厚度和改善上调，但未能营救 超出产后第1天以外的致死性。相反，成人CMS中ATF4的诱导消融对心脏没有影响 功能或左心室维度，在CM发育的特定阶段表明ATF4的不同作用。 综上所述，上述证据使我们提出了以下假设：ATF4在CM中起着至关重要的作用 通过p53依赖性和非依赖性机制的增殖和功能在心脏的特定阶段 发展。因此，我们的具体目的是：1。确定ATF4在心肌细胞中的作用 通过分析ATF4心肌细胞特异性基因敲除（ATF4 CKO）小鼠的增殖和心脏发育，并 阐明ATF4在p53依赖性和/或中调节靶基因途径的机制 - 独立的方式； 2。确定胚胎和新生儿需要哪个ATF4 通过分析ATF4诱导性心肌细胞 - 特定的敲除（ICKO）小鼠。我们提出的研究将帮助我们了解ATF4和 p53以及在体内关键发展阶段的CMS中的其他新型ATF4目标，并确定一个 安全的治疗窗口，用于在心脏病中使用ATF4和/或p53抑制剂的潜在应用。