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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=687993
• 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发现计划的长期目标是以斑马鱼(ZF)为模型，更好地了解硬骨鱼心脏收缩能力的调节机制。这种新颖性在一定程度上源于为解决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、光学标测体外心脏(同时测定膜电位、细胞内钙和心电)、等温滴定量热法、分子动力学模拟、停流光谱荧光法、一系列分子生物学程序(定点突变、重组ZF蛋白表达纯化、qRT-PCR、纳米串mRNA分析和使用CRISPR/Cas9进行基因组编辑)、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，并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感兴趣，因为它们的水生栖息地使它们暴露在陆地哺乳动物没有经历过的水离子浓度、温度和pH的突然和具有挑战性的变化中。鱼可能会随着环境条件的变化而死亡，因为心脏不能充分发挥作用。因此，一个长期目标是了解环境差异是如何决定这些生物体心脏的分子设计的。另一个主要目标是了解环境变化(如温度和pH)如何在分子、细胞、器官和系统水平上显著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生存能力。ZF提供了巨大的优势，可以用来详细了解生理功能的分子调节。像大多数模型一样，它在理解哺乳动物和硬骨鱼的心脏功能方面造成了一些限制，这一点将在提案中得到解决。我们将继续使用在异源表达系统中表达的克隆基因以及在分离的心肌细胞中表达的克隆基因来继续研究硬骨鱼心脏，在这些基因中，我们可以同时测量单个细胞中的力生成、肌节长度、膜电位和[Ca+]i或Phi。在ZF中，我们既可以利用它们基因组的广泛知识，也可以使用基因组编辑工具(例如CRISPR-Cas9)来了解这些不同的钙处理蛋白(包括各种硬骨鱼特有的类似蛋白)在调节收缩能力中的作用。*因此，NSERC的这项研究计划将对硬骨鱼心脏功能调节在一系列重要环境变量上的复杂性提供独特的见解，因为使用了新的但集成的方法，以及研究人员与其合作者的经验。