Functional dissection of the regulatory network that governs cardiomyocyte maturation

控制心肌细胞成熟的调节网络的功能剖析

• 批准号: 10629491
• 负责人: Nathan James VanDusen
• 金额: $ 24.9万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10629491
• 关键词: Adult; Birth; CRISPR screen; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cells; Complex; Congenital Heart Defects; Data; Defect; Deposition; Development; Disease; Disease model; Dissection; Down-Regulation; Epigenetic Process; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Heart; Heart failure; Histone H2B; Homeostasis; Human; Hypertrophic Cardiomyopathy; Hypertrophy; Incidence; Maps; Mentors; Metabolism; Molecular; Morphology; Mus; Mutate; Mutation; Myosin ATPase; Myosin Heavy Chains; Neonatal; Operative Surgical Procedures; Palliative Surgery; Pathologic; Patients; Phase; Phenotype; Process; Production; Protein Isoforms; Protocols documentation; Regenerative Medicine; Regulation; Regulatory Element; Replacement Therapy; Repression; Role; Sarcomeres; Stress; Ubiquitination; chromatin modification; conditional knockout; congenital heart disorder; experimental study; heart function; hemodynamics; histone modification; improved; in vivo; insight; loss of function; molecular targeted therapies; neonate; novel; promoter; regenerative approach; response; sound; targeted treatment; tool; transcription regulatory network

ABSTRACT Between birth and adulthood, cardiomyocytes (CMs) undergo profound changes in size, ultrastructure, metabolism, and gene expression, a process collectively referred to as CM maturation. Although highly coordinated, the transcriptional network that governs this process is not understood. This lack of understanding is a barrier to cardiac regenerative medicine, where our current inability to mature CMs differentiated from non- myocytes limits their use for disease modeling or replacement therapy. In addition, disruption of maturation by abnormal hemodynamic loads in neonates who have undergone surgery to correct congenital heart defects likely contributes to their high incidence of heart failure in adulthood. A sound understanding of the regulatory network governing CM maturation will inspire hypothesis driven attempts to surmount these challenges. In mice, a key hallmark of CM maturation is sarcomere isoform switching, including the well documented neonatal switch from Myosin Heavy Chain 7 (Myh7) to Myosin Heavy Chain 6 (Myh6). We have conducted and validated an in vivo high throughput CRISPR screen for transcriptional regulators of CM maturation, using the Myh7/6 isoform switch as the readout. Two top candidates from this screen, Rnf20 and Rnf40, form a complex which governs deposition of the poorly understood chromatin modification histone-2B mono-ubiquitination (H2Bub1). H2Bub1 function is unexplored in the heart, and associated with congenital heart disease mutations in human patients. Therefore, In Aim 1 we will perform a detailed characterization of the impact of Rnf20/40 on genetic regulation of CM maturation. A major reason why the transcriptional networks controlling CM maturation are poorly understood is that appropriate tools were previously unavailable. In this proposal we will utilize a suite of novel in vivo tools to begin mapping the transcriptional regulatory networks that govern CM maturation. In Aim 2 we will seek to accomplish this goal by dissecting two Myh7 cis-regulatory elements. Furthermore, we will contrast the genetic regulation of CM maturation with pathological hypertrophy by discovering and describing the key regulatory mechanisms of Myh7, which is deactivated during maturation and re-activated in response to pathological stress. These experiments will yield mechanistic clues as to how gene expression is coordinately controlled during maturation and disease, thus enabling improved CM production protocols and targeted therapies. Collectively these experiments will begin to map the transcriptional regulatory network that governs maturation by defining the functions of select trans-acting regulators and cis-regulatory elements.

摘要 从出生到成年，心肌细胞(CM)在大小、超微结构、 代谢和基因表达，这一过程统称为CM成熟。尽管高度 协调一致，管理这一过程的转录网络尚不清楚。这种缺乏理解 是心脏再生医学的障碍，在那里我们目前无法成熟的CMS与非 心肌细胞限制了它们在疾病建模或替代疗法中的使用。此外，成熟的中断是由 先天性心脏病手术后新生儿的异常血流动力学负荷 可能是导致他们成年后心力衰竭发生率高的原因之一。对监管的正确理解 管理CM的网络成熟将激发假说驱动的尝试，以克服这些挑战。 在小鼠中，CM成熟的一个关键标志是肌节异构体转换，包括已有文献记载的 新生儿从肌球蛋白重链7(MyH7)转变为肌球蛋白重链6(MYH6)。我们已经进行了和 验证了体内高通量CRISPR筛选CM成熟的转录调控因子，使用 Myh7/6异构体开关作为读数。这个屏幕上的两个热门候选人RNF20和RNF40组成了一个复合体 它控制着鲜为人知的染色质修饰组蛋白-2B单一泛素化的沉积 (H2 Bub1)。H2Bub1的功能在心脏中是未知的，并与先天性心脏病突变有关 在人类病人身上。因此，在目标1中，我们将详细描述RNF20/40对 CM成熟的遗传调控。 控制CM成熟的转录网络知之甚少的一个主要原因是 以前没有合适的工具。在这项提案中，我们将利用一套新颖的活体工具来 开始绘制管理CM成熟的转录调控网络。在目标2中，我们将寻求 通过解剖两个Myh7顺式调控元件来实现这一目标。此外，我们将对比基因 通过发现和描述关键调控因子来调控CM成熟与病理性肥大 Myh7在成熟过程中失活并在病理反应中重新激活的机制 压力。这些实验将提供基因表达如何协调控制的机制线索。 在成熟期和疾病期间，因此能够改进CM生产方案和靶向治疗。 总的来说，这些实验将开始绘制管理成熟的转录调控网络。 通过定义选定的反式作用调节器和顺式调节器元件的功能。