Regulation of contractility in the teleost heart

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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.