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: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566056
• 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.

NSERC的这项研究计划致力于研究硬骨鱼心脏收缩能力的调节机制，重点是鲑鱼类。硬骨鱼的心肌功能在生理学上引起了极大的兴趣，因为它们的水生栖息地使它们暴露在水离子浓度、水温和pH的突然和具有挑战性的变化中，这是陆地哺乳动物在其环境中所没有经历的。在全球不断变化的环境条件下，鲑鱼等鱼类可能会在某些栖息地死亡，因为心脏不能充分发挥作用。因此，这个研究项目的一个主要目标是了解环境差异是如何决定这些生物心脏的分子设计的。另一个主要目的是了解环境变化(如温度和pH)如何在分子、细胞和系统水平上显著影响硬骨鱼心脏的基本调节机制，从而影响心脏的收缩能力、心输出量和组织生存能力。心脏的收缩能力有很大的变化，但在节拍的基础上受到输送到收缩元件的钙(Ca)的量和/或收缩元件对钙的敏感性的调节。哺乳动物心脏收缩钙的重要来源包括肌浆网(SR)、钙释放通道或兰尼定受体(RyR)的释放和细胞外间隙的内流，该通道主要由肌膜(SL)的L钙通道(CaV1.2)控制。然而，我们认为心脏旁膜钠/钙交换器(NCX1)是钙外流的主要机制，也是调节SL钙内流的主要因素。心肌肌钙蛋白C(CTNC)是目前所检测的所有心脏中细胞内钙升高时启动收缩反应的关键蛋白。因此，本研究计划将利用生物信息学、分子生物学、共聚焦显微镜、生物物理学、成像和生理学等技术，重点研究硬骨鱼心脏中CaV1.2、NCX1、cTNC和RyR的结构和功能。特别是，鲑鱼对我们对心脏收缩的理解构成了一个有趣的挑战，因为它的心肌在人类和大多数哺乳动物心脏停搏的温度范围内(4-15摄氏度)起作用。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，在这些细胞中，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(使用荧光显微镜)。除了鲑鱼外，我们还计划广泛利用我们对其基因组有更详细了解的硬骨鱼模型(例如斑马鱼)，以利用遗传操作来了解这些不同的钙处理蛋白在调节收缩能力中的作用。除了这些了解硬骨鱼心脏调节的细胞和分子方法外，我们还将使用复杂的综合方法。第一种包括光学标测技术，在这种技术中，同时测量心房和心室的膜电位和钙瞬变。这是一项强大的技术，与膜片钳技术相比，它具有非常独特的优势。第二种是光学相干层析成像(OCT)，以高分辨率确定心脏的结构尺寸，包括心腔体积。最后，我们认为，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，将我们的分子和细胞知识整合到系统水平的框架中，这一点很重要。