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.

项目概要 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 ATF/cAMP 反应元件结合 (CREB) 蛋白家族。作为一个井—— ATF4 是一种特有的应激反应转录因子，在低水平下组成型且普遍表达 水平，但可以在各种细胞应激条件下快速诱导。先前的研究表明 ATF4在各种生理和病理条件下在多种细胞类型和组织中发挥作用， 包括心脏病。然而，对于 ATF4 及其靶基因在 哺乳动物心脏发育。为了弥补这一知识差距并确定 ATF4 在 心肌细胞 (CM)，我们生成了新型 Atf4 CM 特异性组成型敲除 (cKO) 和他莫昔芬- 诱导型 CM 特异性敲除 (icKO) 小鼠系。我们的初步研究表明 Atf4 cKO 小鼠 表现出围产期死亡和心脏形态缺陷，与 CM 增殖减少相关。 RNA- 胚胎心室组织的 seq 和 ChIP-seq 分析揭示了一系列细胞周期的上调 已知位于 p53 下游的逮捕相关基因，以及一系列不依赖于 p53 的基因的下调 心脏发育/功能和/或细胞周期进展相关基因，其中大部分含有 ATF4 Atf4 cKO 小鼠中的结合区域。发育中的 CM 中 ATF4 的缺失也会导致 p53 蛋白增加 水平，但不是 Trp53 mRNA 水平。此外，Atf4 cKO 小鼠中的 p53 消融可部分恢复心室壁 厚度和 p53 靶细胞周期停滞基因在 E17.5 上调的改善，但未能挽救 出生后第 1 天后的致死率。相反，成人 CM 中 Atf4 的诱导消融对心脏没有影响 功能或左心室尺寸，表明 ATF4 在 CM 发展的特定阶段具有不同的作用。 综上所述，上述证据使我们得出这样的假设：ATF4 在 CM 中发挥重要作用 在心脏的特定阶段通过 p53 依赖和独立机制进行增殖和功能 发展。因此，我们的具体目标是： 1. 确定 ATF4 在心肌细胞中的作用 通过分析 Atf4 心肌细胞特异性敲除 (Atf4 cKO) 小鼠的增殖和心脏发育，以及 阐明 ATF4 在 p53 依赖性和/或 - 独立的方式； 2. 确定胚胎和新生儿需要 ATF4 的时间 通过 Atf4 诱导型心肌细胞分析心肌细胞增殖和功能（从 E7.5 到 P30） 特异性基因敲除（icKO）小鼠。我们提出的研究将帮助我们了解 ATF4 的具体作用和 p53，以及体内关键发育阶段的 CM 中的其他新型 ATF4 靶标，以及确定 ATF4 和/或 p53 抑制剂在心脏病中潜在应用的安全治疗窗口。