Elucidating the Role of Multinuclearity in Healthy and Diseased Mammalian Cardiomyocytes

项目摘要 心力衰竭抵抗了其他疾病死亡率的下降趋势， needed.心力衰竭是由心肌细胞的肥大和损失驱动的，心肌细胞构成了心脏的大部分。 心脏按体积和提供主要的收缩力，使血液在整个身体。超过 哺乳动物中90%的心肌细胞是多倍体和/或多核的。这些多余的 DNA已经在心脏再生能力低的背景下进行了研究，但相对较少。 努力的重点是这种增加的核性的其他潜在的适应性或适应不良的影响。 从历史上看，成熟心肌细胞的大尺寸已经排除了使用无偏单细胞的分析 RNA测序。研究集中在孤立细胞核中的RNAseq，其本质上缺乏 关于所研究的心脏内的核性的信息。最近，新技术允许无偏见， 完整成熟心肌细胞的高通量单细胞RNAseq。当这些单细胞的结果 研究与孤立核研究相比，研究人员发现，使用相同的聚类， 在一些算法中，不同的细胞转录组簇大约是不同的细胞转录组簇的两倍。 核转录组簇。这一结果表明存在一种形式的“核密码”， 多核细胞的转录组由来自两个或多个核簇的核“编码”， 得到了其他多核细胞群体观察结果的支持。这些编码细胞可以显示 收缩性、抗凋亡性甚至增殖潜力的差异，具有重要意义 心脏功能和心力衰竭的发展。 这位史蒂芬一号的目标是。Katz的建议是：1）利用最先进的单细胞和单核测序 技术来识别和确认这种核编码，2）探索单细胞和多细胞的空间分布， 有核细胞的心脏，和3）确定这种编码如何响应遗传和环境 健康和衰竭的心脏的扰动。根据机制的要求，我们在本提案中提出了一个 计划为我们的实验室的研究一个雄心勃勃的新方向，建立在严格的工作记录在 文献，并得到合作者和主题专家的支持。该提案结合了生物信息学， 成像和下一代测序方法，以确定细胞核密码的变化， 多核心肌细胞导致对心脏损伤和应激反应的差异。完成后， 这项资助将使人们更全面地了解多核性和多倍性在遗传学中的作用。 哺乳动物心脏，随后揭示了许多新的研究途径， 治疗方法来对抗心力衰竭。

PROJECT SUMMARY Heart Failure has resisted the downward trend in mortality seen in other diseases and new therapies are needed. Heart failure is driven by the hypertrophy and loss of cardiomyocytes, which comprise the bulk of the heart by volume and provide the main contractile force necessary to move blood throughout the body. Over 90% of cardiomyocytes in mammalian species are polyploidal and/or multinucleated. These extra copies of DNA have been studied in the context of the low regenerative capacity of the heart, yet comparatively little effort has focused on other potential adaptive or maladaptive effects of this increased nuclearity. Historically, the large size of the mature cardiomyocyte has precluded its analysis using unbiased single cell RNA sequencing. Research has focused on RNAseq in isolated nuclei, which by its very nature lacks information about nuclearity within the studied hearts. Recently, new technology has allowed for unbiased, high-throughput single cell RNAseq of whole, mature cardiomyocytes. When the results of these single cell studies are compared to the isolated nuclei studies, researchers find that, using the same clustering algorithms, there are approximately twice as many distinct cellular transcriptome clusters as there are distinct nuclear transcriptome clusters. This result suggests the existence of a form of ‘nuclear code’ in which a multinucleated cell’s transcriptome is ‘encoded’ by nuclei drawn from two or more nuclear clusters, a result supported by observations from other multinucleated cell populations. These encoded cells could display differences in contractility, resistance to apoptosis, or even proliferative potential, with significant implications for cardiac function and the development of heart failure. The goal of this Steven I. Katz proposal is to 1) Utilize cutting edge single cell and single nucleus sequencing technology to identify and confirm this nuclear encoding, 2) Explore the spatial distribution of mono and multi- nucleated cells across the heart, and 3) Identify how this encoding responds to genetic and environmental perturbations in healthy and failing hearts. As requested by the mechanism, in this proposal we set forth a plan for an ambitious new direction for our laboratory’s research, built upon rigorous work documented in the literature and supported by collaborators and subject matter experts. The proposal combines bioinformatic, imaging, and next generation sequencing approaches to identify how changes in the nuclear code of multinucleated cardiomyocytes leads to differences in response to cardiac injury and stress. Upon completion, this grant will result in a more complete understanding of the role of multinuclearity and polyploidy in the mammalian heart, with subsequent revelation of numerous new avenues of research for diagnostic and therapeutic approaches to combat heart failure.