Regulation of contractility in the teleost heart

硬骨鱼心脏收缩力的调节

• 批准号: RGPIN-2019-07251
• 负责人: Tibbits, Glen
• 金额: $ 2.91万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=739382
• 关键词: Regulation; contractility; teleost; heart

The long-term objective of this NSERC Discovery program is to understand better the mechanisms by which cardiac contractility is regulated in teleosts using zebrafish (ZF) as a model. The novelty, in part, arises from the range and complementarity of complex techniques developed to address the questions being raised. These techniques include: high-resolution echocardiography, optically mapped ex-vivo hearts (determine membrane potential, cytosolic Ca and ECG simultaneously), isothermal titration calorimetry, molecular dynamics simulations, stopped-flow spectrofluorometry, an array of molecular biology procedures (site-directed mutagenesis, recombinant ZF protein expression purification, qRT-PCR, Nanostring mRNA analysis and genome editing using CRISPR/Cas9), cardiomyocyte isolation and patch clamping among others. We believe that this approach is unique in this regard and allows our lab to address important and fundamental questions of teleosts biology in truly novel manner. Myocardial function in teleosts is of great interest physiologically because their aquatic habitat exposes them to sudden and challenging changes in water ion concentrations, temperature and pH that are not experienced by terrestrial mammals. Fish may die with changing environmental conditions because the heart fails to function adequately. Thus, a long-term objective is to understand how environmental differences have dictated the molecular design of the hearts of these organisms. Another major objective is to understand how environmental changes (e.g. temperature and pH) acutely impact the basic regulatory mechanisms in the teleost heart on a molecular, cellular, organ and systems level, thereby impacting cardiac output and organismal viability. The ZF offers tremendous advantages that can be exploited to develop a detailed understanding of the molecular regulation of physiological function. Like most models, it poses some limitations in the understanding of cardiac function in both mammals and teleosts, which will be addressed in the proposal. We will continue to study the teleost heart using cloned genes (that can be easily mutated) expressed in heterologous expression systems as well as in isolated cardiomyocytes in which we can measure force generation, sarcomere length, membrane potential and either [Ca2+]i or pHi simultaneously in a single cell. In ZF we can exploit both extensive knowledge of their genomes as well as the ability to use genome editing tools (e.g. CRISPR-Cas9) to understand the role of these different Ca2+ handling proteins (including a variety of teleost specific paralogs) in regulating contractility. Thus, this NSERC program of research will offer a unique insights into the complexities of the regulation of teleost cardiac function over a range of important environmental variables because of the novel yet integrated approaches being used as well as the experience of the investigator in conjunction with his collaborators.

NSERC Discovery 项目的长期目标是更好地了解以斑马鱼 (ZF) 作为模型的硬骨鱼心肌收缩力的调节机制。新颖性部分源于为解决所提出的问题而开发的复杂技术的范围和互补性。这些技术包括：高分辨率超声心动图、光学映射离体心脏（同时测定膜电位、胞质 Ca 和心电图）、等温滴定量热法、分子动力学模拟、停流荧光光谱法、一系列分子生物学程序（定点诱变、重组 ZF 蛋白表达纯化、 qRT-PCR、使用 CRISPR/Cas9 的 Nanostring mRNA 分析和基因组编辑、心肌细胞分离和膜片钳等。我们相信这种方法在这方面是独一无二的，使我们的实验室能够以真正新颖的方式解决硬骨鱼生物学的重要和基本问题。硬骨鱼的心肌功能在生理学上非常有趣，因为它们的水生栖息地使它们面临水离子浓度、温度和 pH 值的突然且具有挑战性的变化，而陆生哺乳动物不会经历这些变化。由于心脏无法充分发挥功能，鱼可能会随着环境条件的变化而死亡。因此，长期目标是了解环境差异如何决定这些生物体心脏的分子设计。另一个主要目标是了解环境变化（例如温度和 pH 值）如何在分子、细胞、器官和系统水平上严重影响硬骨鱼心脏的基本调节机制，从而影响心输出量和生物体活力。 ZF 具有巨大的优势，可用于详细了解生理功能的分子调节。与大多数模型一样，它对理解哺乳动物和硬骨鱼的心脏功能造成了一些限制，这将在提案中得到解决。我们将继续使用在异源表达系统以及分离的心肌细胞中表达的克隆基因（很容易突变）来研究硬骨鱼心脏，其中我们可以在单个细胞中同时测量力的产生、肌节长度、膜电位以及[Ca2+]i或pHi。在 ZF，我们可以利用其基因组的广泛知识以及使用基因组编辑工具（例如 CRISPR-Cas9）的能力来了解这些不同的 Ca2+ 处理蛋白（包括各种硬骨鱼特异性旁系同源物）在调节收缩性中的作用。 因此，由于所使用的新颖而综合的方法以及研究者与合作者的经验，NSERC 的这项研究计划将为一系列重要环境变量下硬骨鱼心脏功能调节的复杂性提供独特的见解。