Functional characterization of developing heart in embryos using Electric Potential Sensors

The characterization of the electrocardiographic activity of the living zebrafish heart during early developmental stages is a challenging task. Most of the available techniques are limited to heartbeat rate quantification being this inaccurate. Other invasive methodologies require the insertion of electrodes noise isolated environments and advanced amplification stages making these techniques very expensive. In this paper, we present a novel and non-invasive sensor development to characterize the functional activity of the developing heart of in vivo zebrafish embryos. The design is based on the Electric Potential Sensing technology patented at Sussex which has been developed to achieve reproducibility and continuous detection. We present preliminary functional characterization data of the developing zebrafish heart starting at 3 days-post-fertilization. Results show that using the proposed system for mapping the electrocardiographic activity of the zebrafish heart at early developmental stages is successfully accomplished. This is the first time that such a sensitive sensor has been developed for measuring the electrical changes occurring on micron sized (< 100 µm) living samples such as the zebrafish heart

High-resolution 3D optical microscopy inside the beating zebrafish heart using prospective optical gating

3D fluorescence imaging is a fundamental tool in the study of functional and developmental biology, but effective imaging is particularly difficult in moving structures such as the beating heart. We have developed a non-invasive real-time optical gating system that is able to exploit the periodic nature of the motion to acquire high resolution 3D images of the normally-beating zebrafish heart without any unnecessary exposure of the sample to harmful excitation light. In order for the image stack to be artefact-free, it is essential to use a synchronization source that is invariant as the sample is scanned in 3D. We therefore describe a scheme whereby fluorescence image slices are scanned through the sample while a brightfield camera sharing the same objective lens is maintained at a fixed focus, with correction of sample drift also included. This enables us to maintain, throughout an extended 3D volume, the same standard of synchronization we have previously demonstrated in and near a single 2D plane. Thus we are able image the complete beating zebrafish heart exactly as if the heart had been artificially stopped, but sidestepping this undesirable interference with the heart and instead allowing the heart to beat as normal

Dynamic Views of Structure and Function during Heart Morphogenesis

Congenital heart defects remain the most common birth defect in humans, occurring in over 1% of live births. The high prevalence of cardiac malformations can be partially attributed to limited knowledge regarding the embryonic roots of the disease. A variety of congenital heart defects are thought to arise from combinations of genetic and epigenetic factors. In an effort to better understand this dynamic relationship, our study explores the structure and function of the developing heart and valves and examines hemodynamic factors influencing valvulogenesis. In order to study cardiac mechanics, we employed novel high-speed confocal microscopy and four-dimensional visualization techniques. A dynamic four-dimensional dataset describing heart and valve development along with blood flow patterns throughout cardiac morphogenesis is presented. Utilizing newly developed tools, we propose a novel pumping mechanism in the valveless embryonic heart tube via elastic wave propagation and reflection. We show that this form of pumping leads to oscillatory shear stresses in the developing atrio-ventricular canal, a phenomenon that had not previously been documented. An in vivo method to modulate trans-valvular oscillatory flows is described and used to test our hypothesis that oscillatory shear stress across the primitive valve cushions stimulates heart valve leaflet formation. Our results suggest hemodynamic forces contribute to valvulogenesis and enhance our understanding of normal and abnormal heart valve development

Moving Domain Computational Fluid Dynamics to Interface with an Embryonic Model of Cardiac Morphogenesis

Peristaltic contraction of the embryonic heart tube produces time- and spatial-varying wall shear stress (WSS) and pressure gradients (∇P) across the atrioventricular (AV) canal. Zebrafish (Danio rerio) are a genetically tractable system to investigate cardiac morphogenesis. The use of Tg(fli1a:EGFP)y1 transgenic embryos allowed for delineation and two-dimensional reconstruction of the endocardium. This time-varying wall motion was then prescribed in a two-dimensional moving domain computational fluid dynamics (CFD) model, providing new insights into spatial and temporal variations in WSS and ∇P during cardiac development. The CFD simulations were validated with particle image velocimetry (PIV) across the atrioventricular (AV) canal, revealing an increase in both velocities and heart rates, but a decrease in the duration of atrial systole from early to later stages. At 20-30 hours post fertilization (hpf), simulation results revealed bidirectional WSS across the AV canal in the heart tube in response to peristaltic motion of the wall. At 40-50 hpf, the tube structure undergoes cardiac looping, accompanied by a nearly 3-fold increase in WSS magnitude. At 110-120 hpf, distinct AV valve, atrium, ventricle, and bulbus arteriosus form, accompanied by incremental increases in both WSS magnitude and ∇P, but a decrease in bi-directional flow. Laminar flow develops across the AV canal at 20-30 hpf, and persists at 110-120 hpf. Reynolds numbers at the AV canal increase from 0.07±0.03 at 20-30 hpf to 0.23±0.07 at 110-120 hpf (p< 0.05, n=6), whereas Womersley numbers remain relatively unchanged from 0.11 to 0.13. Our moving domain simulations highlights hemodynamic changes in relation to cardiac morphogenesis; thereby, providing a 2-D quantitative approach to complement imaging analysis. © 2013 Lee et al

3D + time blood flow mapping using SPIM-microPIV in the developing zebrafish heart

We present SPIM-μPIV as a flow imaging system, capable of measuring in vivo flow information with 3D micron-scale resolution. Our system was validated using a phantom experiment consisting of a flow of beads in a 50 μm diameter FEP tube. Then, with the help of optical gating techniques, we obtained 3D + time flow fields throughout the full heartbeat in a ∼3 day old zebrafish larva using fluorescent red blood cells as tracer particles. From this we were able to recover 3D flow fields at 31 separate phases in the heartbeat. From our measurements of this specimen, we found the net pumped blood volume through the atrium to be 0.239 nL per beat. SPIM-μPIV enables high quality in vivo measurements of flow fields that will be valuable for studies of heart function and fluid-structure interaction in a range of small-animal models

The Zebrafish (Danio rerio) Is a Relevant Model for Studying Sex-Specific Effects of 17β-Estradiol in the Adult Heart

Cardiovascular diseases are a major cause of morbidity and mortality, and there are significant sex differences therein. However, the underlying mechanisms are poorly understood. The steroid hormone 17β-estradiol (E2) is thought to play a major role in cardiovascular sex differences and to be protective, but this may not hold true for males. We aimed at assessing whether the zebrafish is an appropriate model for the study of E2 effects in the heart. We hypothesized that E2 regulates the cardiac contractility of adult zebrafish in a sex-specific manner. Male and female zebrafish were treated with vehicle (control) or E2 and the cardiac contractility was measured 0, 4, 7 and 14 days after treatment initiation using echocardiography. There was no significant effect on the heart rate by E2. Notably, there was a significant decrease in the ejection fraction of male zebrafish treated with E2 compared with controls. By contrast, there was no major difference in the ejection fraction between the two female groups. The dramatic effect in male zebrafish occurred as early as 4 days post treatment initiation. Although there was no significant difference in stroke volume and cardiac output between the two male groups, these were significantly higher in female zebrafish treated with E2 compared with controls. Gene expression analysis revealed that the levels of estrogen receptors were comparable among all groups. In conclusion, our data demonstrate that the adult zebrafish heart robustly responds to E2 and this occurs in a sex-specific manner. Given the benefits of using zebrafish as a model, new targets may be identified for the development of novel cardiovascular therapies for male and female patients. This would contribute towards the realization of personalized medicine

Optically gated beating-heart imaging

The constant motion of the beating heart presents an obstacle to clear optical imaging, especially 3D imaging, in small animals where direct optical imaging would otherwise be possible. Gating techniques exploit the periodic motion of the heart to computationally "freeze" this movement and overcome motion artefacts. Optically gated imaging represents a recent development of this, where image analysis is used to synchronize acquisition with the heartbeat in a completely non-invasive manner. This article will explain the concept of optical gating, discuss a range of different implementation strategies and their strengths and weaknesses. Finally we will illustrate the usefulness of the technique by discussing applications where optical gating has facilitated novel biological findings by allowing 3D in vivo imaging of cardiac myocytes in their natural environment of the beating heart

Ageing in the zebrafish heart

PhD ThesisWith advancing age there is a progressive decline in the function of the heart. In humans reductions in stroke volume and cardiac output occurs often resulting in cardiac disease and subsequent death. The pathology found in the heart due to advancing age is attributed to a reduction in cardiomyocytes which causes cardiac dysfunction and heart disease, leading to heart failure. Zebrafish are a valuable tool in studying ageing and heart disease. As zebrafish age they gradually senesce. This is similar to humans and other mammals. However the response of the zebrafish heart to ageing has not been explored. The zebrafish heart changes due to ageing, with increased fibrosis and ventricular wall thickness. I have established new assays to measure proliferation and apoptosis in zebrafish cardiomyocytes using multiplexing of thymidine analogues and cleaved caspase 3, respectively. Using these developed assays it was discovered that these changes may be caused by an observed increase in cardiomyocyte apoptosis. This was coupled with no change in cardiomyocyte proliferation. These changes may be mediated by an increase in natriuretic peptide expression. In response to exercise, cardiomyocyte proliferation increases signalled by increased gata4, nkx2.5, tbx5, and mef2c expression and a reduction of natriuretic peptide expression. In the long term these genetic and cellular changes in the heart in response to exercise may slow some of the pathological changes observed in the heart