Identification and analysis of factors that regulate cardiomyocyte maturation

心肌细胞成熟调节因素的鉴定与分析

• 批准号: 9379399
• 负责人: Nathan James VanDusen
• 金额: $ 1.7万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-06 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9379399
• 关键词: Actin-Binding Protein; Actins; Address; Adult; Animals; CRISPR/Cas technology; Candidate Disease Gene; Cardiac; Cardiac Myocytes; Cardiac development; Cell Differentiation process; Cell Size; Cells; ChIP-seq; Clustered Regularly Interspaced Short Palindromic Repeats; Complementary DNA; Complex; Cre-LoxP; Cytoskeleton; Data; Databases; Defect; Development; Dose; Embryo; Employee Strikes; Epigenetic Process; FHOD3 gene; Factor Analysis; Family; Flow Cytometry; GATA4 gene; Gene Family; Genes; Genetic; Genetic Models; Genetic Transcription; Growth; Guide RNA; Heart; Heart Diseases; Individual; Knock-out; Knowledge; Libraries; Link; Maintenance; Mechanics; Mediating; Modeling; Molecular; Morphology; Mosaicism; Mus; Muscle Cells; Mutagenesis; Myocardial; Myocardium; Neonatal; Phase; Phenotype; Play; Pluripotent Stem Cells; Process; Role; Sarcomeres; Somatic Cell; Testing; Time; Tissues; Transcriptional Regulation; Zinc Fingers; base; cost; differential expression; fetal; functional restoration; gain of function; high throughput screening; in vivo; insight; knockout gene; loss of function; member; mutant; novel; overexpression; postnatal; progenitor; regenerative; regenerative therapy; screening; transcription factor; transcriptome sequencing; treatment strategy

Abstract Strategies to restore function in damaged hearts often focus on the replacement of lost cardiomyocytes (CMs). Depending on the approach, the new CMs may be derived through the differentiation of exogenous pluripotent stem cells, the differentiation of resident progenitors, the proliferation of endogenous CMs, or reprogramming of non-CMs to CMs. Unfortunately, CMs generated by these approaches have thus far made poor substitutes for mature myocardium, with deficiencies in both electrical and mechanical function. Studies indicate that CMs from regenerative strategies often resemble CMs of the fetal or neonatal heart, rather than the adult, making CM maturation a major roadblock in the field. This obstacle has been difficult to overcome due to insufficient knowledge of how CM maturation is transcriptionally regulated. Here we propose addressing this deficiency by first utilizing an established genetic model of a CM growth defect to identify and analyze factors that regulate CM maturation. Second, we will combine insights gained from this model with a novel technical approach to screen for novel transcriptional regulators of CM maturation in vivo. GATA4 and GATA6 are zinc finger transcription factors that play key roles in cardiac function and development. Mosaic double knockout of myocardial GATA4/6 via low dose administration of AAV9-TNT-Cre to neonatal mice intriguingly appears to result in stalled CM maturation. By adulthood, GATA4/6 mutant cells are dramatically smaller than their Cre- counterparts, resembling neonatal CMs. As GATA4/6 proved to be crucial and redundant regulators of CM growth, we reasoned that analysis of GATA4 targets is likely to reveal factors that mediate CM maturation. Therefore, we conducted neonatal GATA4 ChIP-seq to identify likely regulators of CM maturation. In Aim 1 we outline a strategy to functionally analyze the role of two promising candidates in vivo during neonatal CM maturation. These factors, Fhod3 and Daam1, belong to the Formin family of actin binding proteins, which have previously been linked to sarcomere assembly and maintenance. The striking phenotype of GATA4/6 mutant CMs indicated that transcriptional regulators of CM maturation can be identified by assessing cell size in a mosaic loss-of-function model. However screening factors in vivo one at a time is prohibitively costly. In Aim 2 we propose an in vivo screen that will utilize cutting edge CRISPR technologies to allow many genes to be tested in a single animal. This screen will use the cell autonomous effect of gene knockout on individual CM growth as the readout. This unbiased approach will be used to discover new transcriptional regulators of neonatal CM growth, which is a hallmark of CM maturation. Successful completion of this Aim will allow us to use candidates as new genetic entry points, which can be exploited by ChIP-seq and RNA-seq to rapidly dissect the transcriptional network that governs neonatal CM growth and maturation. Collectively, the complementary approaches of Aims 1 and 2 will greatly increase our knowledge of this maturation network, and will have the potential to enhance regenerative therapeutic strategies.

抽象的 恢复受损心脏功能的策略通常集中于替换丢失的心肌细胞 （CM）。根据方法的不同，新的 CM 可能是通过外源性物质的分化而产生的。 多能干细胞、常驻祖细胞的分化、内源性 CM 的增殖，或 将非 CM 重新编程为 CM。不幸的是，迄今为止，通过这些方法生成的 CM 已经 成熟心肌的不良替代品，电功能和机械功能均存在缺陷。研究 表明再生策略的 CM 通常类似于胎儿或新生儿心脏的 CM，而不是心脏的 CM 成人，使得 CM 成熟成为该领域的主要障碍。这一障碍一直难以克服，因为 对 CM 成熟如何转录调控的了解不足。在此我们建议解决这个问题 首先利用已建立的 CM 生长缺陷遗传模型来识别和分析因素 调节 CM 成熟。其次，我们将从该模型中获得的见解与新颖的技术结合起来 筛选体内 CM 成熟的新型转录调节因子的方法。 GATA4 和 GATA6 是锌指转录因子，在心脏功能和心脏功能中发挥关键作用。 发展。通过低剂量施用 AAV9-TNT-Cre 对心肌 GATA4/6 进行马赛克双敲除 有趣的是，新生小鼠似乎会导致 CM 成熟停滞。成年后，GATA4/6 突变细胞 比 Cre- 对应物小得多，类似于新生儿 CM。事实证明 GATA4/6 至关重要 以及 CM 生长的冗余调节因子，我们推断对 GATA4 目标的分析可能会揭示一些因素 介导 CM 成熟。因此，我们进行了新生儿 GATA4 ChIP-seq 来鉴定可能的调节因子 CM 成熟。在目标 1 中，我们概述了一种策略，对两种有前途的候选药物在体内的作用进行功能分析 新生儿 CM 成熟期间。这些因子 Fhod3 和 Daam1 属于肌动蛋白结合的福尔明家族 蛋白质，之前已被认为与肌节的组装和维护有关。 GATA4/6 突变体 CM 的显着表型表明 CM 成熟的转录调节因子 可以通过评估镶嵌功能丧失模型中的细胞大小来识别。然而体内筛选因素 一次一个的成本非常高。在目标 2 中，我们提出了一种利用尖端 CRISPR 的体内筛选 允许在单个动物中测试许多基因的技术。此屏幕将使用细胞自主效应 基因敲除对个体 CM 生长的影响作为读数。这种公正的方法将用于发现新的 新生儿 CM 生长的转录调节因子，这是 CM 成熟的标志。顺利完成 这一目标将使我们能够使用候选基因作为新的基因切入点，这些切入点可以被 ChIP-seq 和 RNA-seq 可快速剖析控制新生儿 CM 生长和成熟的转录网络。 总的来说，目标 1 和 2 的互补方法将大大增加我们对此的了解 成熟网络，并将有潜力增强再生治疗策略。

项目成果

Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation.

• DOI: 10.1038/s41467-021-24743-z
• 发表时间: 2021-07-21
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: VanDusen NJ;Lee JY;Gu W;Butler CE;Sethi I;Zheng Y;King JS;Zhou P;Suo S;Guo Y;Ma Q;Yuan GC;Pu WT
• 通讯作者: Pu WT