Despite the widespread use of zebrafish for understanding cardiac development and function, few quantitative tools exist to measure functional cardiovascular phenotypes in embryos. Laser-scanning velocimetry meets this need, using a standard laser-scanning confocal microscope for non-invasive measures of cardiac function. High-resolution ultrasound has been used to measure cardiovascular performance in adult zebrafish , and technically could be applied to the analysis of the zebrafish embryonic cardiovascular system. Unlike confocal microscopes that are available at many research institutions, high-resolution ultrasound requires expensive and highly specialized equipment that are not generally available to many investigators. We have shown that laser-scanning velocimetry provides sensitive and precise measures of cardiac function including contractility, cardiac output, vascular resistance, heart rate, and stroke volume. Our data demonstrate that laser-scanning velocimetry easily measures drug-induced cardiotoxicity, performance changes during cardiovascular development and can diagnose anatomic lesions such as aortic stenosis in genetically-manipulated embryos.
Laser-scanning velocimetry provides quantitative and precise measures of several indices of cardiac performance (summarized in Table 1) and offers significant improvements over existing pre-clinical cardiovascular diagnostic techniques. A recent report by Pan, et. al. combines linescans with correlation spectroscopy to measure the velocity of cells travelling through the dorsal aorta of embryonic zebrafish . While determining velocity vectors by correlation spectroscopy is a significant advance, this technique requires two sequential measurements that severely limit its temporal resolution. Discontinuous measurements of cellular velocities have been made in zebrafish embryos using video microscopy, and in transgenic mouse embryos expressing GFP-labeled erythrocytes using perpendicular linescans [15, 19]. Dual spot cross correlation methods of measuring blood cell velocity have existed since the mid-1970s and should be applicable to measuring blood cell velocity in zebrafish embryos with good temporal resolution [20–23]. These methods use two photodiodes spaced a fixed distance apart along the path of blood flow, and cell velocities are calculated based on the time required for a cell to pass between the two detectors. Because cross-correlation methods only measure the average speed of a cell as it passes between the two detectors, they do not continuously measure velocity. This continuity is required for calculating acceleration and deceleration, which contains important information about contractility and peripheral resistance. The use of a parallel scan line allows us to continuously measure the velocity of the same object so long as it is located within the linear scanning region. This feature is what makes laser-scanning velocimetry unique in its temporal resolution for both velocity and acceleration measurements.
Video microscopy has also been used to measure cardiac output in Xenopus laevis, Gallus gallus, and Danio rerio embryos [11, 12, 24–26]. This technique estimates the volume of blood ejected from the ventricle based on the observed changes in its diameter. Our measurements of cardiac output using laser-scanning velocimetry are in good agreement with these published values . While the cardiac output measurements described here are estimates that assume a circular vessel cross-section, and can be underestimated if linescans are obtained from the aorta after arterial branches, they do not require normal heart morphology and valve function. A more exact determination of cardiac output may be obtained using laser-scanning velocimetry in transgenic zebrafish animals where the vessel dimensions are delineated by an endothelial expressed GFP such as fli1-GFP .
Recently, the genes responsible for two mutant zebrafish embryos that have similar cardiovascular phenotypes, santa and valentine, were mapped to ccm1 and ccm2, respectively . The valentine mutant contains a mutation in OSM and exhibits pericardial edema, a dilated heart, and a complete lack of blood flow . Morpholino knockdown of OSM resulted in significantly reduced circulation relative to control zebrafish. The fact that morpholino knockdown of OSM did not completely inhibit blood flow is probably a result of low level OSM expression in contrast to complete loss of functional OSM protein in the valentine mutant.
The observation that OSM plays a role in aortic arch lumen formation has important implications for the pathobiology of cerebral cavernous malformations. Mice homozygous for a gene-trap insertion of β-galactosidase into the ccm1/Krit1 locus also have a constricted aortic arch , indicating that a loss of Krit1 in mouse phenocopies a loss of OSM in zebrafish. When viewed in light of evidence that OSM and Krit1 physically associate with each other at sites of actin reorganization , these data suggest that Krit1 and OSM may act as a complex to organize developmental signals required for proper aortic arch lumen formation.
Zebrafish are a popular model for drug screens because of their genetic similarity to humans, the speed of screening in a physiological context and cost-effectiveness . In addition to providing a diagnostic tool for molecular geneticists, laser-scanning velocimetry has obvious applications in pre-clinical drug development. For example, laser-scanning velocimetry could easily be used to identify anti-arhythmic compounds capable of reversing the fibrillating phenotype of the tremblor or reggae mutants . Similarly, the potency and efficacy of potential inotropic drugs, which increase the force of ventricular contractions in heart failure, could easily be screened in wild-type zebrafish embryos to find those compounds that best increase the peak acceleration of blood cells. As demonstrated in Figure 2, in addition to supporting pre-clinical efficacy studies, laser-scanning velocimetry can also be used to readily identify molecules that possess cardiotoxicity.
Laser-scanning velocimetry quantifies the effects of genetic and pharmacological modifiers of cardiovascular function. With the increasing popularity of zebrafish in basic science and the pharmaceutical industry, the development of additional techniques to correlate physiological function with genes and their chemical modifiers promises to accelerate drug discovery and development in the post-genomic area.