Regulation of cardiomyocyte proliferation by the Reptin ATPase

Reptin ATP酶对心肌细胞增殖的调节

• 批准号: 10747229
• 负责人: Felicia E Wranitz
• 金额: $ 3.56万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2025-09-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10747229
• 关键词: ATAC-seq; ATP phosphohydrolase; Adult; Birth; Bromodeoxyuridine; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cause of Death; Cell Cycle; Cell Cycle Arrest; Cell division; Chromatin; Cicatrix; Complex; Congenital Heart Defects; DNA; DNA Damage; DNA Repair; DNA-dependent ATPase; Data; Data Set; Development; Dissection; Dominant-Negative Mutation; Down-Regulation; Embryo; Engineering; Failure; Family member; Feedback; Fertilization; Gamma-H2AX; Gene Expression; Gene Expression Regulation; Genes; Genetic Models; Genetic Transcription; Heart; Heart Diseases; Heart Injuries; Human; Hyperplasia; Infarction; Injury; Learning; Life; Mediating; Messenger RNA; Modeling; Molecular; Morbidity - disease rate; Mutation; Myocardial; Myocardial Infarction; Myocardium; Natural regeneration; PRC1 Protein; Pathway interactions; Phenotype; Proliferating; Publishing; Regulation; Reporting; Repression; Resistance; Role; S-Phase Fraction; Siblings; Signal Transduction; Source; TP53 gene; Testing; Therapeutic; Transcript; Transcription Factor AP-1; Transgenes; Up-Regulation; Zebrafish; cardiac regeneration; cardiac repair; cardiogenesis; chromatin remodeling; cohort; comparison control; congenital heart disorder; cyclin A2; experimental study; healing; heart damage; insight; loss of function; loss of function mutation; mortality; mutant; novel; novel therapeutics; overexpression; postnatal; regenerative; regenerative therapy; transcriptome sequencing

ABSTRACT: Cardiomyocyte proliferation is an important source of new myocardium during heart development and regeneration. After birth cardiomyocytes are highly resistant to proliferation. The lack of adult cardiomyocyte proliferation precludes cardiac repair and underlies the high morbidity and mortality rates associated with congenital heart defects and adult cardiovascular disease. Therefore, identifying novel regulators of cardiomyocyte proliferation is key to the development of regenerative heart therapies. Zebrafish are a well- established model to study cardiac development and regeneration because their cardiomyocytes maintain a remarkable capacity to proliferate allowing the heart to regenerate after adult injury. Our lab recently reported that the AAA+ ATPase Reptin is a potent suppressor of cardiomyocyte proliferation. Reptin is a known component of the Tip60 and INO80 complexes which have roles in DNA damage repair and chromatin remodeling. Our lab demonstrated in zebrafish that reptin loss of function mutations cause myocardial hyperplasia at 3 days post fertilization (dpf). We showed that cardiomyocyte-specific overexpression of reptin rescues the cardiomyocyte hyperproliferation phenotype. We further demonstrated that inducible overexpression of reptin after adult cardiac injury resulted in decreased cardiomyocyte proliferation and failure to regenerate. Expanding on our published data, I propose to study the molecular mechanisms by which Reptin suppresses cardiomyocyte proliferation. Preliminary data suggests that the ATPase function of Reptin is essential to dampen proliferation at 3 dpf. RNA sequencing of reptin mutant and control cardiomyocytes at 3 dpf revealed the unanticipated result that the hyperproliferative reptin mutant cardiomyocytes upregulate both pro-proliferative (e.g. fosl1a & junba) and anti-proliferative (e.g. tp53 & cbx7a) transcripts. Analysis at 4 and 5 dpf revealed that the reptin mutant cardiomyocytes lose their hyperproliferative phenotype by 4 dpf and have significantly reduced proliferation rates compared to controls at 5 dpf. I hypothesize: 1) that reptin mutant cardiomyocytes require AP-1 activity for their initial proliferative burst where DNA damage accumulates and triggers tp53 upregulation to halt cell division, and 2) that compensatory upregulation of the PRC1 complex component cbx7a drives an anti-proliferative chromatin landscape in reptin mutant cardiomyocytes. In Aim 1, I will assess the sufficiency of AP-1 family members to drive cardiomyocyte proliferation in embryonic zebrafish, the accumulation of DNA damage in hyperproliferative reptin mutant cardiomyocytes, and the role of tp53 upregulation in DNA damage signaling and cell cycle arrest in reptin mutant hearts. In Aim 2, I will identify changes in the chromatin landscape that are associated with reptin loss of function and correlate those alterations to changes in gene expression. I will also investigate the ability of cbx7a to dampen cardiomyocyte proliferation in wildtype and reptin mutant backgrounds. The proposed studies will give insight into the regulation of cardiomyocyte proliferation and identify druggable pathways/targets for novel regenerative therapies.

摘要：心肌细胞增殖是心脏发育过程中新生心肌的重要来源 和再生。出生后的心肌细胞对增殖具有高度抵抗性。缺乏成年心肌细胞 增殖阻碍了心脏修复，并且是与心脏病相关的高发病率和死亡率的基础。 先天性心脏病和成人心血管疾病。因此，鉴定新的调节剂， 心肌细胞增殖是开发再生心脏疗法的关键。斑马鱼是一种- 建立了研究心脏发育和再生的模型，因为他们的心肌细胞保持着 在成年人受伤后，它具有非凡的增殖能力，使心脏能够再生。我们的实验室最近报告说 AAA+ ATP酶Reptin是心肌细胞增殖的有效抑制剂。雷普廷是一位 Tip 60和INO 80复合物的组分，其在DNA损伤修复和染色质中起作用 重塑我们的实验室在斑马鱼中证明，reptin功能丧失突变导致心肌梗死。 在受精后3天（DPF）增生。我们发现心肌细胞特异性的reptin过度表达， 挽救心肌细胞过度增殖表型。我们进一步证明了诱导型过表达 在成人心脏损伤后给予reptin导致心肌细胞增殖减少和再生失败。 在我们已发表的数据的基础上，我建议研究Reptin抑制 心肌细胞增殖初步数据表明，Reptin的ATP酶功能对于抑制 在3dpf时增殖。reptin突变体和对照心肌细胞在3dpf的RNA测序显示， 这是一个意料之外的结果，过度增殖的reptin突变型心肌细胞上调了促增殖和促凋亡蛋白表达。 (e.g. Fosl 1a和Junba）和抗增殖（例如TP 53和CBX 7a）转录物。在4和5 dpf的分析显示， reptin突变心肌细胞在4dpf时失去其过度增殖表型 在5dpf与对照相比的增殖率。我假设：1）reptin突变心肌细胞需要 AP-1活性用于其初始增殖爆发，其中DNA损伤累积并触发tp 53 上调以停止细胞分裂，和2）PRC 1复合物补偿性上调 组分CBX 7A在reptin突变心肌细胞中驱动抗增殖染色质景观。在 目的1，评估AP-1家族成员是否足以驱动胚胎心肌细胞增殖， 斑马鱼，过度增殖的reptin突变心肌细胞中DNA损伤的积累，以及 tp 53在reptin突变心脏的DNA损伤信号传导和细胞周期阻滞中的上调。在目标2中，我将确定 与reptin功能丧失相关的染色质景观的变化， 基因表达的改变。我还将研究cbx 7a抑制心肌细胞增殖的能力， 在野生型和reptin突变体背景中的增殖。拟议的研究将深入了解监管情况 的心肌细胞增殖，并确定药物途径/新的再生疗法的目标。