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.

抽象的 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，这个过程统称为 CM 成熟。虽然高度 协调一致，控制这一过程的转录网络尚不清楚。这种不理解 是心脏再生医学的一个障碍，我们目前无法将成熟的 CM 与非再生医学区分开来。 肌细胞限制了它们在疾病建模或替代治疗中的用途。此外，成熟的破坏 接受先天性心脏病矫正手术的新生儿血流动力学负荷异常 可能导致他们成年后心力衰竭的高发病率。对监管有充分的了解 网络管理 CM 的成熟将激发假设驱动的尝试来克服这些挑战。 在小鼠中，CM 成熟的一个关键标志是肌节亚型转换，包括有据可查的 新生儿从肌球蛋白重链 7 (Myh7) 转换为肌球蛋白重链 6 (Myh6)。我们已经进行并 验证了 CM 成熟转录调节因子的体内高通量 CRISPR 筛选，使用 Myh7/6 同工型开关作为读数。此屏幕中的两个顶级候选者 Rnf20 和 Rnf40 形成一个复合体 它控制着人们知之甚少的染色质修饰组蛋白 2B 单泛素化的沉积 （H2Bub1）。 H2Bub1 在心脏中的功能尚未被探索，并且与先天性心脏病突变有关 在人类患者中。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 以前没有适当的工具。在本提案中，我们将利用一套新颖的体内工具来 开始绘制控制 CM 成熟的转录调控网络。在目标 2 中，我们将寻求 通过剖析两个 Myh7 顺式调控元件来实现这一目标。此外，我们将对比遗传 通过发现和描述关键调节来调节 CM 成熟与病理性肥大 Myh7 的机制，它在成熟过程中失活并在响应病理学时重新激活 压力。这些实验将产生关于如何协调控制基因表达的机制线索 成熟和疾病期间，从而能够改进 CM 生产方案和靶向治疗。 总的来说，这些实验将开始绘制控制成熟的转录调控网络 通过定义选定的反式作用调节因子和顺式调节元件的功能。