Mechanisms of regulating cardiomyocyte chromatin dynamics and regenerative transitions by Tbx20

项目摘要/摘要 心血管疾病是世界上主要的死亡原因，成年人在心脏病发作后康复的能力 心肌细胞(CMS)的静止状态阻碍了心肌梗死(MI)等损伤。轶事 有证据表明，青春期前的人类可以通过体内CMS的增殖来补充损伤后的CMS。 胚胎和新生小鼠也可以在损伤后再生CMS，但再生潜力在第一次损伤后丧失 出生后一周。通过激活Robust促进成年小鼠CMS再生增殖的尝试失败 细胞周期效应因子导致病理性肥大和心功能下降。相比之下，我们的实验室 证明了对胎儿CM增殖至关重要的转录因子Tbx20的条件诱导- 在不损害正常和心肌梗死后成人CMS的情况下促进胎儿特征和抑制增殖 心脏功能。虽然这一发现具有巨大的治疗意义，但其背后的机制是 未知，特别是因为Tbx20在CM成熟和再生过渡过程中起作用 目前定义不明确。此外，越来越多的证据表明Tbx20与SWI/SNF染色质相互作用 重塑成分BRG1促进胎儿和胎儿细胞周期活性相关基因的表达 新生儿CMS，为Tbx20的基因调控作用提供了另一个方面。我们假设Tbx20 与BRG1合作通过重塑CM促进成人CMS的增殖和胎儿特征 染色质进入更不成熟的、类似胎儿的状态，并通过打开与胎儿有关的染色质区 Tbx20转录靶点。我们提出了两个不同的目的来阐明Tbx20的机制 促进成年CMS的增殖和胎儿返胎。 在目标1中，将利用整合的多组学方法来确定Tbx20如何动态调节基因 在正常的CM发育和再生过渡过程中的表达，包括其结合靶点和 染色质变化的影响(目标1.1)。我们将使用以下方法来表征这种再生表型的可逆性 Tbx20感应的暂态模型(目标1.2)。这将决定增殖性、胎儿型CMS是否可以 在去除Tbx20后成熟为成年CMS，这是适应新陈代谢所必需的 成年人内心的需求。在目标2中，我们将利用免疫共沉淀和芯片定量聚合酶链式反应来阐明 Tbx20和BRG1之间的相互作用发生在未成熟的CMS中(Aim 2.1)。我们还将使用条件 小鼠心肌梗死后Tbx20诱导和BRG1缺失模型确定Tbx20诱导是否促进 增殖、胎儿特征和通过BRG1依赖机制改善心功能(目标2.2)。 了解CM成熟过程中的基因调控机制将促进治疗策略 旨在重新激活胎儿基因程序，促进心肌再生，改善心肌梗死后心功能。 这项提案的目的是在Tbx20的背景下阐明这一点，BRG1是一个潜在的新合作伙伴 在正常发育和再生修复期间指导胎儿基因程序。

Project Summary/Abstract Cardiovascular disease is the leading cause of death in the world, and an adult’s ability to recover after a cardiac injury, such as myocardial infarction (MI) is hindered by the quiescent state of cardiomyocytes (CMs). Anecdotal evidence suggests that pre-adolescent humans can replenish CMs after injury by proliferation of resident CMs. Fetal and neonatal mice can also regenerate CMs after injury, but regenerative potential is lost beyond the first postnatal week. Failed attempts to promote regenerative proliferation in adult mouse CMs by activating robust cell cycle effectors have led to pathological hypertrophy and reduced cardiac function. In contrast, our laboratory demonstrated that conditional induction of Tbx20- a transcription factor critical for fetal CM proliferation- promotes fetal characteristics and restrained proliferation in normal and post-MI adult CMs without impairing cardiac function. While this finding has enormous therapeutic relevance, the mechanism underlying this is unknown, particularly because Tbx20 function throughout CM maturation and regenerative transitions are currently ill-defined. Moreover, increasing evidence suggests that Tbx20 interacts with the SWI/SNF chromatin remodeling component BRG1 to promote the expression of genes related to cell cycle activity in fetal and neonatal CMs, providing another facet to the gene regulatory actions of Tbx20. We hypothesize that Tbx20 cooperates with BRG1 to promote proliferation and fetal characteristics in adult CMs by remodeling CM chromatin into a more immature, fetal-like state, and by opening chromatin regions associated with fetal Tbx20 transcriptional targets. We propose two separate Aims to elucidate the mechanism by which Tbx20 promotes proliferation and fetal reversion in adult CMs. In Aim 1, will utilize an integrative multiomics approach to identify how Tbx20 dynamically regulates gene expression throughout normal CM development and regenerative transitions, including its binding targets and effect on chromatin changes (Aim 1.1). We will characterize the reversibility of this regenerative phenotype using a transient model of Tbx20 induction (Aim 1.2). This will determine whether proliferative, fetal-like CMs can mature into adult CMs following the removal of Tbx20, which is necessary to accommodate the metabolic demands of the adult heart. In Aim 2, we will utilize co-immunoprecipitation and CHIP-qPCR to elucidate whether an interaction between Tbx20 and BRG1 occurs in immature CMs (Aim 2.1). We will also utilize conditional mouse models of Tbx20 induction and Brg1 deletion to determine whether Tbx20 induction after MI promotes proliferation, fetal characteristics, and improved cardiac function via a BRG1-dependent mechanism (Aim 2.2). Understanding gene regulatory mechanisms during CM maturation will advance therapeutic strategies aimed at reactivating fetal gene programs, promoting CM regeneration, and improving cardiac function post-MI. The Aims in this proposal seek to elucidate this in the context of Tbx20, with BRG1 as a potential novel partner in directing the fetal gene program during normal development and regenerative repair.