Abstract
ABSTRACTZebrafish are increasingly used as a vertebrate model to study human cardiovascular disorders. Although heart structure and function are readily visualized in zebrafish embryos because of their optical transparency, the lack of effective tools for evaluating the hearts of older, nontransparent fish has been a major limiting factor. The recent development of high-frequency echocardiography has been an important advance for in vivo cardiac assessment, but it necessitates anesthesia and has limited ability to study acute interventions. We report the development of an alternative experimental ex vivo technique for quantifying heart size and function that resembles the Langendorff heart preparations that have been widely used in mammalian models. Dissected adult zebrafish hearts were perfused with a calcium-containing buffer, and a beat frequency was maintained with electrical stimulation. The impact of pacing frequency, flow rate and perfusate calcium concentration on ventricular performance (including end-diastolic and end-systolic volumes, ejection fraction, radial strain, and maximal velocities of shortening and relaxation) were evaluated and optimal conditions defined. We determined the effects of age on heart function in wild-type male and female zebrafish, and successfully detected hypercontractile and hypocontractile responses after adrenergic stimulation or doxorubicin treatment, respectively. Good correlations were found between indices of cardiac contractility obtained with high-frequency echocardiography and with the ex vivo technique in a subset of fish studied with both methods. The ex vivo beating heart preparation is a valuable addition to the cardiac function tool kit that will expand the use of adult zebrafish for cardiovascular research.
Highlights
The zebrafish (Danio rerio) has emerged as a popular vertebrate model for cardiovascular research (Gut et al, 2017; Stoyek et al, 2016)
Establishment of an ex vivo perfusion system To enable quantification of cardiac pump function in beating explanted adult zebrafish hearts, we developed an ex vivo perfusion system adapted from our previously established method of isolating membrane-intact zebrafish cardiomyocytes (Dvornikov et al, 2014), which was based on recommendations from the study by Zhang et al (2011)
Hearts were quickly isolated from anesthetized adult zebrafish, most of the atrium was excised, the atrioventricular canal was exposed, and the ventricle was cannulated with a 34G catheter that was microsurgically sutured to the atrium close to the atrioventricular canal
Summary
The zebrafish (Danio rerio) has emerged as a popular vertebrate model for cardiovascular research (Gut et al, 2017; Stoyek et al, 2016). Despite having only 2 chambers, zebrafish hearts have striking physiological similarities to human hearts (Becker et al, 2011; Werdich et al, 2012) and extensive genetic conservation (Bakkers, 2011; Howe et al, 2013). Optically transparent zebrafish embryos have been mainly used to study heart development and congenital heart disease. The potential utility of zebrafish for study of adult-onset heart diseases is relatively underexplored. Several studies provided proof-of-principle evidence for the value of zebrafish models for assessing the functional effects of human genetic variants (Asimaki et al, 2014; Huttner et al, 2013; Reischauer et al, 2014; Yang et al, 2016). In addition to studies using embryonic zebrafish models (Bendig et al, 2006; Hassel et al, 2009; Knoll et al, 2007), studies have used adult zebrafish to find new cardiomyopathy genes through mutagenesis screening (Ding et al, 2013, 2016) and for evaluation of therapeutic strategies such as mammalian target of rapamycin inhibition (Ding et al, 2011, 2012)
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