Lamin B2 regulates nuclear remodeling in cardiomyocyte terminal differentiation

Lamin B2 调节心肌细胞终末分化中的核重塑

• 批准号: 10579284
• 负责人: Bernhard Kuhn
• 金额: $ 32.76万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10579284
• 关键词: ATAC-seq; Adolescent; Adult; Apoptosis; Birth; Cardiac Myocytes; Cell Cycle; Cell Death; Cell Nucleus; Cells; Cessation of life; Childhood; Chromatin; Chromatin Structure; Chronic; Communication; Cytoplasm; Development; Diameter; Disease; Event; Gene Expression; Gene Expression Profiling; Gene Silencing; Gene Transfer; Genes; Genetic Transcription; Growth; Health; Heart; Heart Diseases; Heart Injuries; Heart failure; Human; Injury; Interdisciplinary Study; Intermediate Filaments; Ischemia; Lead; Mammals; Mitosis; Modeling; Molecular; Morbidity - disease rate; Mus; Myocardial; Natural regeneration; Neonatal; Nuclear; Nuclear Import; Nuclear Lamina; Nuclear Pore; Outcome; Patients; Ploidies; Polyploidy; Predisposition; Progressive Disease; Proliferating; Public Health; Regenerative Medicine; Regenerative capacity; Reperfusion Therapy; Research; Resistance; Role; Signal Transduction; Signaling Molecule; Specificity; Techniques; Testing; Viral; Withdrawal; Work; cardiac regeneration; cdc Genes; gain of function; heart function; in vivo; innovation; knockout gene; lamin B2; loss of function; mortality; mouse model; myocardial injury; neonatal mice; new therapeutic target; novel therapeutics; nucleocytoplasmic transport; single-cell RNA sequencing; superresolution microscopy; transcriptome sequencing

ABSTRACT Heart disease is the leading cause of morbidity and mortality worldwide. Different types of heart injury lead to the development of heart failure, a chronically progressive disease. Heart failure is characterized by increased cardiomyocyte death and insufficient regeneration of new ones. As such, increasing cardiomyocyte regeneration and decreasing death represent targets for new therapies. This application is for developing a transformative paradigm connecting these important cellular mechanisms with nuclear remodeling in cardiomyocyte differentiation. Our new results show that when cardiomyocytes differentiate they decrease the number of nuclear pores (NP). NP are large channels of > 100 nm outer diameter for communication between the nucleus and the cytoplasm. NP, along with the nuclear lamina (NL), function in regulating the nuclear transport of signaling molecules and chromatin organization. Although heart failure alters nuclear transport, the structural and functional changes of NL and NP changes during terminal differentiation are unknown. To determine the molecular mechanisms directing the decrease of NP, we have used single-cell transcriptional profiling, which identified a decrease in expression of lamin B2 (Lmnb2), an intermediate filament and component of the NL, during cardiomyocyte differentiation. Our new results show that Lmnb2 gene knockout in cardiomyocytes blocks M-phase, that is, nuclei do not divide, and instead become polyploid. In addition, the M-phase block decreases NP incorporation. As a result, although the DNA contents of nuclei (ploidy) increases, the number of NP decreases by 50%. The lower NP number identifies a central event in nuclear remodeling, as it indicates not only altered nuclear transport, but also altered chromatin structure. Together, this could explain the decreased ability of terminally differentiated cardiomyocytes to activate cell cycle genes and their increased susceptibility to cell death. This proposal aims to develop a new mechanistic paradigm of nuclear remodeling in cardiomyocyte differentiation, which is synergistic with recent advances in characterizing chromatin changes. We will test the central hypothesis that decreased Lmnb2 gene expression is a central mechanism of nuclear remodeling in cardiomyocyte differentiation. We have assembled an interdisciplinary research team and prepared innovative techniques (super-resolution microscopy, single-cell RNAseq, ATACseq) that, combined with cardiomyocyte-specific Lmnb2flox inactivation and viral expression of Lmnb2, will enable us to determine its role in nuclear remodeling in cardiomyocyte differentiation. The anticipated results will enable future research toward understanding and targeting nuclear remodeling in myocardial development, regeneration, and disease. This will be broadly significant for patients with congenital and acquired heart diseases.

摘要 心脏病是世界范围内发病率和死亡率的主要原因。不同类型的心脏损伤会导致 心力衰竭是一种慢性进行性疾病。心力衰竭的特征是 心肌细胞死亡和新生心肌再生不足。因此，增加心肌细胞 再生和减少死亡是新疗法的目标。此应用程序用于开发 将这些重要的细胞机制与核重塑联系起来的变革性范式 心肌细胞分化。 我们的新结果表明，当心肌细胞分化时，它们减少了细胞核的数量 毛孔(Np)。NP是外径为100 nm的大通道，用于核和核之间的通信 细胞质。NP与核膜(NL)一起在调节信号的核运输方面起作用 分子和染色质组织。尽管心力衰竭改变了核运输，但结构和 NL和NP在终末分化过程中的功能变化尚不清楚。要确定 指导NP减少的分子机制，我们使用了单细胞转录图谱，这是 确定层蛋白B2(Lmnb2)的表达减少，Lmnb2是NL的中间丝和成分， 在心肌细胞分化过程中。我们的新结果表明，Lmnb2基因在心肌细胞中被敲除 阻断M期，也就是细胞核不分裂，而变成多倍体。此外，M相块 减少NP的掺入。因此，虽然细胞核(倍体)的DNA含量增加，但 NP下降50%。较低的NP数字标识了核重构的中心事件，正如它所表明的那样 不仅改变了核运输，还改变了染色质结构。总而言之，这可以解释 终末分化心肌细胞激活细胞周期基因的能力降低和增强 对细胞死亡易感性。 这一建议旨在发展一种新的心肌细胞核重塑的机制范式。 分化，这与最近在表征染色质变化方面的进展是协同的。我们将测试 中心假说认为Lmnb2基因表达减少是核的中心机制 心肌细胞分化中的重塑。我们已经组建了一个跨学科的研究团队， 准备了创新技术(超分辨率显微镜、单细胞RNAseq、ATACseq)，结合 通过心肌细胞特异性Lmnb2flx的失活和Lmnb2的病毒表达，将使我们能够确定其 核重塑在心肌细胞分化中的作用。 预期的结果将使未来的研究能够理解和瞄准核 心肌发育、再生和疾病中的重塑。这将对患者具有广泛的意义 患有先天性和获得性心脏病。