Regulation of cardiomyocyte proliferation by the Reptin ATPase

摘要：心肌细胞增殖是心脏发育过程中新心肌的重要来源 和再生。出生后的心肌细胞对增殖具有高度抵抗力。成人心肌细胞的缺乏 增殖妨碍心脏修复，并导致与以下疾病相关的高发病率和死亡率 先天性心脏病和成人心血管疾病。因此，确定新的调节器 心肌细胞增殖是再生心脏疗法发展的关键。斑马鱼是一口井- 建立了研究心脏发育和再生的模型，因为它们的心肌细胞维持着 非凡的增殖能力，使心脏在成人受伤后能够再生。我们的实验室最近报告说 AAA+ATPase Reptin是一种有效的心肌细胞增殖抑制因子。雷普丁是一位众所周知的 在DNA损伤修复和染色质中起作用的Tip60和INO80复合体的组成 改建。我们的实验室在斑马鱼身上证明了瑞普汀功能突变的丧失会导致心肌 受精后3天(DPF)出现增生。我们发现了心肌细胞特异性过表达的Reptin 挽救心肌细胞过度增殖表型。我们进一步证明了可诱导的过度表达 成人心脏损伤后瑞普汀的应用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，我提议研究Reptin抑制的分子机制 心肌细胞增殖。初步数据表明，Reptin的ATPase功能对于抑制 在3dpf时增殖。Reptin突变体和对照心肌细胞在3DPF时的RNA测序显示 令人意想不到的结果是，高增殖的瑞普汀突变的心肌细胞上调了促增殖 (例如，fosl1a和juba)和抗增殖基因(例如，tp53和cbx7a)转录本。在4和5 DPF下的分析表明 Reptin突变的心肌细胞在4dpf后失去了其高增殖表型，并显著减少 在5DPF时，增殖率与对照组相比。我假设：1)瑞普汀突变的心肌细胞需要 AP-1在其最初的增殖爆发中的活性，其中DNA损伤累积并触发TP53 上调以停止细胞分裂，以及2)PRC1复合体的代偿性上调 组分cbx7a在瑞普汀突变的心肌细胞中驱动抗增殖染色质景观。在……里面 目的1、评价AP-1家族成员在胚胎心肌细胞增殖中的作用。 斑马鱼，在高增殖的瑞普汀突变的心肌细胞中DNA损伤的积累，以及 突变心脏中TP53上调DNA损伤信号和细胞周期停滞。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 基因表达变化的改变。我还将研究cbx7a抑制心肌细胞的能力。 野生型和瑞普汀突变背景下的增殖。拟议的研究将使人们深入了解该法规。 对心肌细胞增殖的研究，并确定新的再生疗法的可用药途径/靶点。

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.