Regulation of contractility in the teleost heart

NSERC发现项目的长期目标是以斑马鱼（ZF）为模型，更好地理解硬骨鱼调节心脏收缩力的机制。其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、光学成像离体心脏（同时测定膜电位、胞质钙和心电图）、等温滴定量热法、分子动力学模拟、停止流动荧光光谱法、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、纳米链mRNA分析和使用CRISPR/Cas9进行基因组编辑）、心肌细胞分离和膜片夹紧等。我们相信这种方法在这方面是独一无二的，并允许我们的实验室以真正新颖的方式解决硬骨鱼生物学的重要和基本问题。

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.