Regulation of contractility in the teleost heart

硬骨鱼心脏收缩力的调节

• 批准号: RGPIN-2014-03724
• 负责人: Tibbits, Glen
• 金额: $ 2.48万
• 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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=666833
• 关键词: Regulation; contractility; teleost; heart

This NSERC research program addresses the mechanisms by which cardiac contractility is regulated in teleosts with a focus on salmonids. Myocardial function in teleosts is of great interest physiologically because their aquatic habitat exposes them to sudden and challenging changes in water ion concentrations, water temperature and pH that are not experienced by terrestrial mammals in their environments. Fish such as salmonids may die in certain habitats with globally changing environmental conditions because the heart fails to function adequately. Thus a major objective in this program of research 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 and systems level, thereby impacting cardiac contractility, cardiac output and organismal viability.* Cardiac contractility is quite variable but is regulated on a beat-to-beat basis by the amount of calcium (Ca) delivered to the contractile element and/or the sensitivity of the contractile element to Ca. Critical sources of contractile Ca in the mammalian heart include release from the sarcoplasmic reticulum (SR) Ca release channel or ryanodine receptor (RyR) and influx from the extracellular space which is controlled primarily by the L-type Ca channel (CaV1.2) in the sarcolemma (SL). However, we believe that the cardiac paralog of the Na+/Ca2+ exchanger (NCX1) while being the primary mechanism of Ca efflux from the myocyte and is also a major player in the regulation of SL Ca influx. Cardiac troponin C (cTnC) is the critical protein that initiates contraction in response to elevated cytosolic Ca in all hearts examined to date. Thus this program of research will focus on the structure and function of CaV1.2, NCX1, cTnC and RyR in the teleost heart using techniques from bioinformatics, molecular biology, confocal microscopy, biophysics, imaging and physiology. In particular, the salmonids pose an interesting challenge to our understanding of cardiac contraction as its myocardia function over a range of temperatures (4-15oC) that are cardioplegic to humans and most mammals. 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 [Ca]i or pHi (using fluorescence microscopy) simultaneously in a single cell. In addition to the salmonids, we plan to make extensive use of model teleosts in which we have more detailed knowledge of their genomes (e.g. zebrafish) in order to exploit the use of genetic manipulations to understand the role of these different Ca handling proteins in regulating contractility.* In addition to these cellular and molecular approaches to understanding teleost cardiac regulation, we will also use sophisticated integrated approaches. The first includes optical mapping techniques in which membrane potential and Ca transients are measured in both the atria and the ventricles, simultaneously. This is a powerful technique which offers very distinctive advantages over patch clamping individual myocytes. The second is optical coherence tomography (OCT) to determine cardiac structural dimensions including chamber volumes with high resolution. Lastly we feel it is important that we are able to use ultra-high frequency echocardiography to study the function of these hearts in the intact fish (including zebrafish) to integrate our molecular and cellular knowledge into a systems level framework.

这个NSERC的研究计划解决的机制，心脏收缩力是调节硬骨鱼与重点鲑鱼。 硬骨鱼类的心肌功能在生理上具有极大的兴趣，因为它们的水生栖息地使它们暴露于水离子浓度、水温和pH值的突然和具有挑战性的变化，而陆生哺乳动物在其环境中不会经历这些变化。 鱼类，如鲑鱼，可能会在全球环境条件变化的某些栖息地死亡，因为心脏无法充分发挥作用。 因此，这项研究计划的一个主要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，从而影响心脏收缩力，心输出量和生物体活力。 心脏收缩力是相当可变的，但在心跳到心跳的基础上通过递送到收缩元件的钙（Ca）的量和/或收缩元件对Ca的敏感性来调节。哺乳动物心脏收缩性Ca的关键来源包括肌浆网（SR）Ca释放通道或ryanodine受体（RyR）的释放和主要由肌膜（SL）中的L型Ca通道（CaV1.2）控制的细胞外间隙的内流。然而，我们认为，心脏的Na+/Ca 2+交换器（NCX 1）的异常是Ca流出的主要机制，也是SL Ca内流的主要调节者。 心肌肌钙蛋白C（cTnC）是迄今为止检查的所有心脏中响应于升高的胞浆Ca而启动收缩的关键蛋白。 因此，这项研究计划将重点关注CaV1.2，NCX 1，cTnC和RyR在硬骨鱼心脏中的结构和功能，使用生物信息学，分子生物学，共聚焦显微镜，生物物理学，成像和生理学技术。 特别是，鲑鱼对我们理解心脏收缩提出了一个有趣的挑战，因为它的心肌功能在一个温度范围内（4- 15 ℃），对人类和大多数哺乳动物都是心脏停搏。 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]i或pHi（使用荧光显微镜）同时在一个单一的细胞。 除了鲑鱼，我们计划广泛使用模型硬骨鱼，我们对它们的基因组有更详细的了解（例如斑马鱼），以便利用遗传操作来了解这些不同的Ca处理蛋白在调节收缩性中的作用。 除了这些细胞和分子的方法来了解硬骨鱼心脏调节，我们还将使用复杂的综合方法。 第一种包括光学标测技术，其中同时测量心房和心室中的膜电位和Ca瞬变。 这是一种强大的技术，它提供了非常独特的优势，膜片钳个别肌细胞。 第二种是光学相干断层扫描（OCT），以确定心脏结构尺寸，包括高分辨率的腔室容积。 最后，我们认为重要的是，我们能够使用超高频超声心动图来研究完整鱼类（包括斑马鱼）中这些心脏的功能，以将我们的分子和细胞知识整合到系统水平的框架中。