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.

摘要 恢复受损心脏功能的策略通常集中在替换丢失的心肌细胞 （CM）。根据方法的不同，新的CM可以通过分化外源的 多能干细胞的分化、固有祖细胞的分化、内源性CM的增殖，或 将非CM重编程为CM。不幸的是，这些方法产生的CM到目前为止 成熟心肌的不良替代品，具有电和机械功能的缺陷。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 成年，使CM成熟在该领域的主要障碍。这一障碍一直难以克服， 对CM成熟是如何转录调控的了解不足。在此，我们建议解决这一问题。 通过首先利用CM生长缺陷的已建立的遗传模型来识别和分析因素， 调节CM成熟的基因。第二，我们将联合收割机从这个模型中获得的见解与一种新的技术相结合， 方法筛选新的转录调控CM成熟在体内。 GATA 4和GATA 6是锌指转录因子，在心脏功能中起关键作用， 发展通过低剂量施用AAV 9-TNT-Cre对心肌GATA 4/6进行嵌合双敲除， 有趣的是，新生小鼠似乎导致CM成熟停滞。到成年时，GATA 4/6突变细胞 明显小于Cre-对应物，类似于新生儿CM。由于GATA 4/6被证明是至关重要的， 和CM生长的冗余调节因子，我们推断GATA 4靶点的分析可能揭示了 介导CM成熟。因此，我们进行了新生儿GATA 4 ChIP-seq，以确定可能的调节因子， CM成熟。在目标1中，我们概述了一种策略，以功能分析两个有前途的候选人在体内的作用 在新生儿CM成熟期间。这些因子Fhod 3和Daam 1属于肌动蛋白结合的形成蛋白家族 这些蛋白质以前与肌节组装和维持有关。 GATA 4/6突变CM的显著表型表明CM成熟的转录调控因子 可以通过评估马赛克功能丧失模型中的细胞大小来识别。然而体内筛选因素 一次一个的成本太高了。在Aim 2中，我们提出了一种利用尖端CRISPR的体内筛选方法， 技术允许在一只动物身上测试许多基因。此屏幕将使用细胞自主效果 基因敲除对个体CM生长的影响作为读数。这种无偏见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。成功完成 这一目标的实现将使我们能够使用候选人作为新的遗传入口点，这可以通过ChIP-seq和 RNA-seq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和目标2的互补办法将大大增加我们对此的了解 成熟网络，并将有潜力提高再生治疗策略。

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