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上 (CMS)。根据不同的方法，新的细胞质雄性不育可能是通过分化外源不育而产生的 多能干细胞，常驻祖细胞的分化，内源性CMS的增殖，或 将非CMS重新编程为CMS。不幸的是，到目前为止，通过这些方法产生的CMS已经 成熟心肌替代物差，电功能和机械功能均有缺陷。研究 表明来自再生策略的CMS通常类似于胎儿或新生儿心脏的CMS，而不是 成年，使CM成熟成为该领域的主要障碍。这一障碍很难克服，因为 对CM成熟如何转录调控知之甚少。在这里，我们建议解决这个问题 缺陷，首先利用已建立的CM生长缺陷的遗传模型来识别和分析因素 来调节CM的成熟。其次，我们将把从该模型中获得的见解与一种新的技术相结合 筛选CM体内成熟新的转录调控因子的方法。 GATA4和GATA6是锌指转录因子，在心脏功能和 发展。小剂量AAV9-TNT-Cre对心肌GATA4/6嵌合型双基因敲除的研究 耐人寻味的是，新生的小鼠似乎导致CM成熟停滞。成年后，GATA4/6突变细胞 比CRE对应的要小得多，类似于新生儿CMS。因为GATA4/6被证明是至关重要的 和多余的CM增长调节器，我们推断对GATA4目标的分析可能会揭示因素 它调节着CM的成熟。因此，我们进行了新生儿GATA4芯片序列分析，以确定可能的调控因子。 Cm成熟。在目标1中，我们概述了一种策略，从功能上分析两个有希望的候选者在活体中的作用 在新生儿CM成熟过程中。这些因子Fhod3和Daam1属于肌动蛋白结合的Forin家族 蛋白质，以前被认为与肌节的组装和维护有关。 GATA4/6突变体CMS的显著表型表明，CM的转录调控因子成熟 可以通过在马赛克功能丧失模型中评估细胞大小来确定。然而，体内的筛选因素 一次一个，成本高得令人望而却步。在目标2中，我们提出了一种活体筛查，它将利用尖端的CRISPR 允许在一只动物身上测试许多基因的技术。此屏幕将使用细胞自主效果 以基因敲除对个体CM生长的影响为读数。这种不偏不倚的方法将被用于发现新的 新生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将候选基因用作新的基因入口点，这可以被Chip-seq和 RNA-seq来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这方面的了解。 成熟网络，并将具有加强再生治疗策略的潜力。

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