Regulation of contractility in the teleost heart

这个NSERC发现计划的长期目标是更好地了解以斑马鱼（ZF）为模型的硬骨鱼类动物心脏收缩力调节的机制。 这种新奇性部分来自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，光学标测离体心脏（同时测定膜电位、胞浆Ca和ECG）、等温滴定量热法、分子动力学模拟、停流荧光分光光度法、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、Nanostring mRNA分析和使用CRISPR/Cas9的基因组编辑），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。硬骨鱼类的心肌功能在生理上具有极大的意义，因为它们的水生栖息地使它们暴露于陆生哺乳动物所没有经历的水离子浓度、温度和pH值的突然和具有挑战性的变化。 鱼类可能会因环境条件的变化而死亡，因为心脏无法充分发挥功能。 因此，一个长期的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提供了巨大的优势，可以利用这些优势来详细了解生理功能的分子调控。 像大多数模型一样，它对哺乳动物和硬骨鱼类的心脏功能的理解造成了一些限制，这将在提案中得到解决。 我们将继续研究硬骨鱼心脏使用克隆的基因（可以很容易地突变）在异源表达系统中表达，以及在分离的心肌细胞中，我们可以测量力的产生，肌节长度，膜电位和[Ca 2 +]i或pHi同时在一个单一的细胞。 在ZF中，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Ca 2+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，NSERC的研究计划将提供一个独特的见解，在一系列重要的环境变量下调节硬骨鱼心脏功能的复杂性，因为使用了新颖而综合的方法，以及研究人员与合作者的经验。

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.**