Physiology 2016, Joint Meeting of the American Physiological Society and The Physiological Society, 29 – 31 July 2016, Convention Centre Dublin, Ireland

3D heart cell culture model from Zebrafish larvae for cardiac research

NAMES: Jake Ireland1, Holly Shiels1, Lisa Mohamet2, Bianka Grunow3

Affiliation:1 Faculty of Life Sciences, 2School of Dentistry, Faculty of Human & Medical Science, University of Manchester. 3 University of Greifswald, Zoological Institute and Museum.

Zebrafish (ZF) provide good models for human cardiac arrhythmias with comparable action potentials and high sequence identity to humans for many ion channels. Previous studies showed that ZF cardiomyocytes spontaneously propagate from embryonic progenitor cells to a mature 3D myocardium in vitro, termed Zebrafish heart aggregates (ZFHAs). Although generated from ZF larvae, ZFHAs show similar cellular architecture to adult ZF in terms of sarcomere structures and cell-to-cell connections. Although, past studies have assessed the contribution of adrenergic stimulation in the electrophysiology of adult and larval ZF, little is known about ZFHAs. This studies aim was to distinguish the role of cardiac adrenergic receptor function in regulating contraction frequency (CF) and inotropic response in larval ZF compared to ZFHAs. To help further validate ZFHAs as an in vitro cardiac model we assessed distinct differences in ECG pattern, CF and size under adrenergic stimulation with phenylephrine. Observations were compared to control ZFHA under normal culture conditions in high glucose DMEM. To generate ZFHAs, whole ZF larvae were homogenized in accordance with schedule 1 methods required by the home office. Cardiac function was observed using an inverted microscope with a high-speed camera. CF was determined by measuring the time interval necessary for 30 heart beats and size of aggregates was measured using ImageJ. Due to lack of consistent morphology in ZFHAs, inotropic patterns were determined using chromogenic software developed in R to analyze differences in perimeter movements. Control ZFHAs show gradual decrease in size and increase in CF between 1-6 days post-homogenization (d.p.h) day1(Size=8191.9±1083.1𝜇𝑚$, CF=45±3BPM) day6(Size=7614.6±1428.3𝜇𝑚$, CF= 67±12BMP) while phenylephrine caused increased trends in size and CF compared to control, day1(Size=6252.4±958.7𝜇𝑚$, CF=52±14BPM) day6(Size=9048.4±6829.3𝜇𝑚$ CF= 75±22BMP). At 13d.p.h, a positive chronotropic change was observed in ZFHAs supplemented with phenylephrine (p=0.0026, n=12), which aligned with a significant increase in size seen at 12d.p.h (p=0.0017, n=12). Finally, phenylephrine caused a significant (p=0.0415, n=74) positive correlation between size and CF (r=0.2376) while control had no correlation between CF and size (p=0.3333, n=253). Chromogenic patterns of ZFHAs in response to phenylephrine showed positive inotropic responses and more defined ECG patterns. This suggests that ZFHA have similar developmental patterns of adrenergic receptors to larval ZF. It is also observed that response to the agonist phenylephrine results in positive chronotropic and inotropic tone that maintains CF and size beyond 6d.p.h. Response of ZFHAs to adrenergic stimulation could highlight important correlations between human and ZFHA response to chronotropic drugs developed for cardiac arrhythmias that focus on adrenergic manipulation.

24th Northern Cardiovascular Research Group Meeting Leeds University, 16th April 2016

3D heart cell culture model from Zebrafish larvae for cardiac research

Authors: Jake Ireland1, Patricia Hodgson1, Holly Shiels1, Lisa Mohamet2, BiankaGrunow3

Affiliation:1 Faculty of Life Sciences, 2School of Dentistry, Faculty of Human & Medical Science, University of Manchester. 3 University of Greifswald, Zoological Institute and Museum.

Development of anti-arrhythmic drugs requires in-depth knowledge of ion channels and their contribution to the action potential and cardiac rhythm. Development of new drugs is a slow and expensive process resulting in an ever-increasing demand for novel, high-throughput and cost-effective compound screening assays. Compared to other models Zebrafish (ZF) provide an excellent platform for modelling human cardiac diseases since they have comparable action potential rhythms and have high sequence identity to a number of ion channels. Previous studies showed that fish heart cells can be spontaneously propagated from embryonic heart progenitor cells to a mature 3D myocardium in vitro, termed Zebrafish heart aggregates (ZFHAs). In this study we report on beat rate, size, cellular architecture and ECG patterns over a series of developmental time points. We have refined our model and improved yield and consistency of ZFHAs by maintaining energy homeostasis with the addition of lactate to the culture media. Comparing different homogenization techniques of embryonic tissue with bead blasting, pestle & mortar and slow collagenase-vortex digestion has also helped improve consistency. Understanding the electrophysiology changes and hypertrophic responses of these ZFHAs to beta-adrenergic agonists, will help us better assess their potential use as screening assays.

The Heart – New Horizons 24th February 2016 CTF Building, University of Manchester.

Generating an in vitro 3D heart cell culture model from zebrafish larvae for cardiac research 

Patricia Hodgson1 & Jake Ireland1, Holly Shiels1*, Lisa Mohamet2, Bianka Grunow3

1School of Medicine, Faculty of Life Sciences, University of Manchester 2School of Dentistry, Faculty of Human & Medical Science, University of Manchester. 3 University of Greifswald, Zoological Institute and Museum * Email: holly.shiels@manchester.ac.uk


 

Genetics and Biochemistry 3rd Year Showcase – 27 March 2015, Grove Building Swansea University

Posters

Genomic and phenotypic comparison of Escherichia coli isolates from host and environmental sources

Jake Ireland, Guillaume Méric, Leonardos Mageiros, Samuel K. Sheppard Medical Microbiology and Infectious Disease, College of Medicine, Swansea University, UK

Medical Microbiology and Infectious Disease, College of Medicine, Swansea University, UK