recovery-of-adult-zebrafish-hearts-for-high-throughput-applications

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

University of California San Francisco

Long QT syndrome (LQTS) is a disorder of ventricular repolarization that predisposes affected individuals to lethal cardiac arrhythmias. To date, an appropriate animal model of inherited LQTS does not exist. The zebrafish is a powerful vertebrate model used to dissect molecular pathways of cardiovascular development and disease. Because fundamental electrical properties of the zebrafish heart are remarkably similar to those of the human heart, the zebrafish may be an appropriate model for studying human inherited arrhythmias. Here we describe the molecular, cellular, and electrophysiological basis of a zebrafish mutant characterized by ventricular asystole. Genetic mapping and direct sequencing identify the affected gene as kcnh2, which encodes the channel responsible for the rapidly activating delayed rectifier K(+) current (I(Kr)). We show that complete loss of functional I(Kr) in embryonic hearts leads to ventricular cell membrane depolarization, inability to generate action potentials (APs), and disrupted calcium release. A small hyperpolarizing current restores spontaneous APs, implying wild-type function of other ionic currents critical for AP generation. Heterozygous fish manifest overt cellular and electrocardiographic evidence for delayed ventricular repolarization. Our findings provide insight into the pathogenesis of homozygous kcnh2 mutations and expand the use of zebrafish mutants as a model system to study human arrhythmias.

Zebrafish model for human long QT syndrome.

Vertebrate hearts depend on highly specialized cardiomyocytes that form the cardiac conduction system (CCS) to coordinate chamber contraction and drive blood efficiently and unidirectionally throughout the organism. Defects in this specialized wiring system can lead to syncope and sudden cardiac death. Thus, a greater understanding of cardiac conduction development may help to prevent these devastating clinical outcomes. Utilizing a cardiac-specific fluorescent calcium indicator zebrafish transgenic line, Tg(cmlc2:gCaMP)(s878), that allows for in vivo optical mapping analysis in intact animals, we identified and analyzed four distinct stages of cardiac conduction development that correspond to cellular and anatomical changes of the developing heart. Additionally, we observed that epigenetic factors, such as hemodynamic flow and contraction, regulate the fast conduction network of this specialized electrical system. To identify novel regulators of the CCS, we designed and performed a new, physiology-based, forward genetic screen and identified for the first time, to our knowledge, 17 conduction-specific mutations. Positional cloning of hobgoblin(s634) revealed that tcf2, a homeobox transcription factor gene involved in mature onset diabetes of the young and familial glomerulocystic kidney disease, also regulates conduction between the atrium and the ventricle. The combination of the Tg(cmlc2:gCaMP)(s878) line/in vivo optical mapping technique and characterization of cardiac conduction mutants provides a novel multidisciplinary approach to further understand the molecular determinants of the vertebrate CCS.

Genetic and physiologic dissection of the vertebrate cardiac conduction system.

Much of the effort in understanding the dynamic process of development has focused on dissecting biochemical pathways. Recent studies illustrate that simple physical forces are also important in patterning organs.

Developmental biology: physics adds a twist to gut looping.

Dilated cardiomyopathy is a leading cause of congestive heart failure and a debilitating complication of antineoplastic therapies. Despite disparate causes for dilated cardiomyopathy, maladaptive cardiac remodeling and decreased systolic function are common clinical consequences, begging an investigation of in vivo contractile dynamics in development and disease, one that has been impossible to date.

Actin Binding GFP Allows 4D In Vivo Imaging of Myofilament Dynamics in the Zebrafish Heart and the Identification of Erbb2 Signaling as a Remodeling Factor of Myofibril Architecture.

Article	Year
Zebrafish model for human long QT syndrome. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 17592134	2007
Genetic and physiologic dissection of the vertebrate cardiac conduction system. PLoS biology\| PubMed ID: 18479184	2008
Developmental biology: physics adds a twist to gut looping. Current biology : CB\| PubMed ID: 22032191	2011
Actin Binding GFP Allows 4D In Vivo Imaging of Myofilament Dynamics in the Zebrafish Heart and the Identification of Erbb2 Signaling as a Remodeling Factor of Myofibril Architecture. Circulation research\| PubMed ID: 25228389	2014

Rima Arnaout

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