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 心脏病是全球发病率和死亡率的主要原因。不同类型的心脏损伤导致 心力衰竭是一种慢性进行性疾病。心力衰竭的特征是 心肌细胞死亡和新细胞再生不足。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一个 将这些重要的细胞机制与细胞核重塑联系起来的变革范式， 心肌细胞分化 我们的新结果表明，当心肌细胞分化时， 孔（NP）。NP是外径> 100 nm的大通道，用于细胞核和细胞核之间的通信。 细胞质NP与核纤层（NL）沿着，调节信号的核转运 分子和染色质组织。虽然心力衰竭改变了核运输，但结构和 在终末分化过程中NL和NP的功能变化是未知的。确定 为了研究指导NP减少的分子机制，我们使用了单细胞转录谱， 确定了核纤层蛋白B2（Lmnb2）表达的减少，核纤层蛋白B2是NL的中间丝和组分， 在心肌细胞分化过程中。我们的新结果表明，心肌细胞中的Lmnb2基因敲除 阻断M期，即细胞核不分裂，而是变成多倍体。此外，M相块 降低NP掺入。结果，虽然细胞核的DNA含量（倍性）增加，但细胞核的数目增加。 NP下降了50%。较低的NP数确定了核重塑的中心事件，因为它表明 不仅改变了核转运，而且改变了染色质结构。综合起来，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， 对细胞死亡的敏感性。 本研究旨在建立一种新的心肌细胞核重构机制 分化，这是协同与表征染色质变化的最新进展。我们将测试 中心假设，Lmnb2基因表达减少是核分裂的中心机制， 心肌细胞分化中的重塑。我们组建了一个跨学科的研究团队， 准备创新技术（超分辨率显微镜，单细胞RNAseq，ATACseq），结合 心肌细胞特异性Lmnb2flox失活和Lmnb2的病毒表达，将使我们能够确定其 在心肌细胞分化中的核重塑中的作用。 预期的结果将使未来的研究能够了解和瞄准核武器。 重塑在心肌发育、再生和疾病中的作用。这将对患者具有广泛意义 先天性和后天性心脏病患者