Regulation of contractility in the teleost heart

这个NSERC研究项目解决了硬骨鱼的心脏收缩力受到调节的机制，重点是鲑鱼。硬骨鱼的心肌功能在生理学上有很大的兴趣，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值的突然和具有挑战性的变化中，而这些变化是陆地哺乳动物在其环境中所没有经历过的。在全球环境条件不断变化的某些栖息地，鲑鱼等鱼类可能会死亡，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、心输出量和有机体活力。心脏的收缩力是可变的，但在搏动的基础上受到钙（Ca）传递到收缩元件的量和/或收缩元件对Ca的敏感性的调节。哺乳动物心脏中可收缩的钙的关键来源包括从肌浆网（SR）释放Ca释放通道或ryanodine受体（RyR）以及主要由肌膜（SL）中的l型Ca通道（CaV1.2）控制的细胞外空间的内流。然而，我们认为心脏的Na+/Ca2+交换器（NCX1）虽然是钙从肌细胞外排的主要机制，但也是调节SL钙内流的主要参与者。心肌肌钙蛋白C （cTnC）是一种重要的蛋白，在所有心脏中，当细胞内钙升高时，它会引发收缩。因此，本项目将利用生物信息学、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提出了一个有趣的挑战，因为它的心肌功能在温度范围内（4-15摄氏度），这对人类和大多数哺乳动物来说是心脏瘫痪的。我们将继续研究硬骨鱼心脏，使用在异源表达系统中表达的克隆基因（可以很容易地突变），以及在分离的心肌细胞中，我们可以在单个细胞中同时测量力产生、肌节长度、膜电位和[Ca]i或pHi（使用荧光显微镜）。除了鲑鱼，我们计划广泛使用模型硬骨鱼，其中我们有更详细的基因组知识（例如斑马鱼），以便利用基因操作来了解这些不同的钙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理解硬骨鱼心脏调节，我们也将使用复杂的综合方法。第一种包括光学测绘技术，其中膜电位和钙瞬态同时在心房和心室测量。这是一项强大的技术，与贴片夹紧单个肌细胞相比，它提供了非常独特的优势。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体积。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这些心脏在完整鱼类（包括斑马鱼）中的功能，将我们的分子和细胞知识整合到系统级框架中。

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.