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: 10372035
• 负责人: Bernhard Kuhn
• 金额: $ 38.22万
• 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 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372035
• 关键词: ATAC-seq; Adolescent; Adult; Apoptosis; Birth; Caliber; Cardiac Myocytes; Cell Cycle; Cell Death; Cell Nucleus; Cells; Cessation of life; Childhood; Chromatin; Chromatin Structure; Chronic; Communication; Cytoplasm; Development; 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; Microscopy; 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; Public Health; Regenerative Medicine; Regenerative capacity; Reperfusion Therapy; Research; Resistance; Resolution; Role; Signal Transduction; Signaling Molecule; Specificity; Techniques; Testing; Viral; Withdrawal; Work; cardiac regeneration; cardiogenesis; 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; 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.

抽象的 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 心力衰竭的发展，一种长期进行性疾病。心力衰竭的特征是增加 心肌细胞死亡和新的再生不足。因此，增加心肌细胞 再生和死亡减少代表新疗法的靶标。该应用程序用于开发一个 将这些重要的细胞机制与核重塑连接起来的变化范式 心肌细胞分化。 我们的新结果表明，当心肌细胞区分时，它们会减少核的数量 孔（NP）。 NP是> 100 nm外径的大通道，用于在细胞核之间通信 细胞质。 NP与核层（NL）一起在调节信号传导的核转运方面发挥作用 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在终端分化过程中的功能变化尚不清楚。确定 指导NP减少的分子机制，我们使用了单细胞转录分析， 鉴定出lamin B2（LMNB2）的表达降低，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，心肌细胞中的LMNB2基因敲除 块M期，即核不分裂，而是变成多倍体。另外，M相块 减少NP掺入。结果，尽管核的DNA含量（倍性）增加，但数量 NP降低50％。 NP较低的数字标识了核重塑的中心事件，因为它表明 不仅改变了核转运，还改变了染色质结构。一起，这可以解释 终末分化的心肌细胞激活细胞周期基因的能力降低了，它们的增加 对细胞死亡的敏感性。 该建议旨在开发心肌细胞中核重塑的新机械范式 分化，这是表征染色质变化的最新进展。我们将测试 降低LMNB2基因表达是核的中心假设 心肌细胞分化中的重塑。我们组建了一个跨学科研究团队， 准备的创新技术（超分辨率显微镜，单细胞RNASEQ，atacseq），合并 随着心肌细胞特异性LMNB2Flox灭活和LMNB2的病毒表达，将使我们确定其 在心肌细胞分化中核重塑中的作用。 预期的结果将使未来的研究能够理解和靶向核 重塑心肌发育，再生和疾病。这对于患者来说是显着意义的 与先天性和心脏病有关。