Abstract
ABSTRACTThe zebrafish (Danio rerio) is an increasingly popular model organism in cardiovascular research. Major insights into cardiac developmental processes have been gained by studies of embryonic zebrafish. However, the utility of zebrafish for modeling adult-onset heart disease has been limited by a lack of robust methods for in vivo evaluation of cardiac function. We established a physiological protocol for underwater zebrafish echocardiography using high frequency ultrasound, and evaluated its reliability in detecting altered cardiac function in two disease models. Serial assessment of cardiac function was performed in wild-type zebrafish aged 3 to 12 months and the effects of anesthetic agents, age, sex and background strain were evaluated. There was a varying extent of bradycardia and ventricular contractile impairment with different anesthetic drugs and doses, with tricaine 0.75 mmol l−1 having a relatively more favorable profile. When compared with males, female fish were larger and had more measurement variability. Although age-related increments in ventricular chamber size were greater in females than males, there were no sex differences when data were normalized to body size. Systolic ventricular function was similar in both sexes at all time points, but differences in diastolic function were evident from 6 months onwards. Wild-type fish of both sexes showed a reliance on atrial contraction for ventricular diastolic filling. Echocardiographic evaluation of adult zebrafish with diphtheria toxin-induced myocarditis or anemia-induced volume overload accurately identified ventricular dilation and altered contraction, with suites of B-mode, ventricular strain, pulsed-wave Doppler and tissue Doppler indices showing concordant changes indicative of myocardial hypocontractility or hypercontractility, respectively. Repeatability, intra-observer and inter-observer correlations for echocardiographic measurements were high. We demonstrate that high frequency echocardiography allows reliable in vivo cardiac assessment in adult zebrafish and make recommendations for optimizing data acquisition and analysis. This enabling technology reveals new insights into zebrafish cardiac physiology and provides an imaging platform for zebrafish-based translational research.
Highlights
The zebrafish (Danio rerio) is an increasingly popular vertebrate model organism for research studies into human diseases because of their low maintenance costs, high fecundity, fast generation times and ease of genetic manipulation (Bakkers, 2011; Shih et al, 2015).Embryonic zebrafish are highly informative for investigating cardiac developmental processes owing to the optical transparency of young fish that allows direct visualization of the heart
With regard to sex differences, we found that indexing to weight, body mass index (BMI) and body surface area (BSA) abolished differences in end-diastolic volume (EDV) between male and female fish of the same age, whereas indexing to length did not
diphtheria toxin A (DTA)-mediated cardiomyocyte loss impairs ventricular contractile function To determine whether pathological myocardial contractile impairment could be effectively identified by echocardiography, we evaluated cardiac function in male wild-type fish (n=8) before and 7 days after induction of DTA by 4-hydroxy-tamoxifen (4-HT) (Fig. 5; Tables S6, S7; Movies 4, 5)
Summary
The zebrafish (Danio rerio) is an increasingly popular vertebrate model organism for research studies into human diseases because of their low maintenance costs, high fecundity, fast generation times and ease of genetic manipulation (Bakkers, 2011; Shih et al, 2015).Embryonic zebrafish are highly informative for investigating cardiac developmental processes owing to the optical transparency of young fish that allows direct visualization of the heart. Adult zebrafish have been shown to develop profound ventricular remodeling in response to environmental insults (Hein et al, 2015; Sun et al, 2009). These important observations point to the untapped potential of adult zebrafish for studying a broad range of human heart disorders including heritable and acquired cardiomyopathies and postinfarction myocardial regeneration. Heart rate, ventricular dimensions and cardiac function were compared in male fish (n=10-13) from four separate wild-type strains (TE, SUN, WIK, AB), each aged 9 months. All images were analyzed using FIJI ImageJ version 1.48k (National Institutes of Health, https://imagej.nih.gov/ij/)
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