High-resolution cardiovascular function confirms functional orthology of myocardial contractility pathways in zebrafish

Abstract

Phenotype-driven screens in larval zebrafish have transformed our understanding of the molecular basis of cardiovascular development. Screens to define the genetic determinants of physiological phenotypes have been slow to materialize as a result of the limited number of validated in vivo assays with relevant dynamic range. To enable rigorous assessment of cardiovascular physiology in living zebrafish embryos, we developed a suite of software tools for the analysis of high-speed video microscopic images and validated these, using established cardiomyopathy models in zebrafish as well as modulation of the nitric oxide (NO) pathway. Quantitative analysis in wild-type fish exposed to NO or in a zebrafish model of dilated cardiomyopathy demonstrated that these tools detect significant differences in ventricular chamber size, ventricular performance, and aortic flow velocity in zebrafish embryos across a large dynamic range. These methods also were able to establish the effects of the classic pharmacological agents isoproterenol, ouabain, and verapamil on cardiovascular physiology in zebrafish embryos. Sequence conservation between zebrafish and mammals of key amino acids in the pharmacological targets of these agents correlated with the functional orthology of the physiological response. These data provide evidence that the quantitative evaluation of subtle physiological differences in zebrafish can be accomplished at a resolution and with a dynamic range comparable to those achieved in mammals and provides a mechanism for genetic and small-molecule dissection of functional pathways in this model organism.

Fig. 1.

Measurement of cardiovascular function in zebrafish. A: developing zebrafish embryo at 3 days post fertilization (dpf). The zebrafish is a vertebrate model organism that is transparent during early development. The zebrafish heart and circulation are easily visible with light microscopy. The heart consists of a single atrium and ventricle that lie anteriorly on the ventral surface of the fish (A, left box) To identify a reproducible location for analysis of circulation, the aorta adjacent to the cloaca (A, right box) was chosen as a defined imaging landmark. The heart and area adjacent to the cloaca are shown in higher magnification in B and D. A brightfield image (B) and illustration (C) show the important anatomy of the zebrafish ventricle. The ventricle is oriented so that it empties rostrally with each contraction through the bulbus arteriosus (BA). At the ventral side of the fish the pericardium lies adjacent to the myocardium on the outer curvature of the heart, with the yolk lying adjacent to the inner curvature of the ventricle. The ventricular cavity (red, C) is readily identified by the blood within it. The cloaca identified a consistent landmark for microscopy to examine flow in the dorsal aorta (D and E, oriented with the rostrum to the left). Individual erythrocytes could be visualized as they transited the artery. Image analysis tools allowed the delineation of the endomyocardial border (red polygon, F) either at end systole or diastole to define the ventricular area or to place manually a scan line across the midventricular short axis (green line). The image intensity across the pixels of the scan line was then measured (G), stored in an array (materials and methods), and converted into an M-mode image (H). Yellow bar, 50 μm; white bar, 250 ms; blue bars, short-axis end-diastolic (D_d) and end-systolic (D_s) dimensions. Myocardial thickness at end diastole (MT_d) and end systole (MT_s) were also measured (brackets). Bar in D = 100 μm.

Fig. 2.

Measurement of ventricular size and function in vtn^−/− mutants. Brightfield image of zebrafish ventricles from wild-type (wt; A1) and vtn^−/− (B1) hearts in lateral projection and M mode of wt (A2) and vtn^−/− (B2) ventricles. C: short- and long-axis ventricular diameter in wt and vtn^−/− hearts. D: fractional shortening (FS) measured in wt and vtn^−/− hearts. SAx, short axis. E–G: ventricular area, ventricular performance index [fractional area change (FAC)], and ventricular volume compared between wt and vtn^−/− ventricles. All data are expressed as means ± SE for n = 9 measurements per condition. Differences in which P < 0.05 are denoted in diastole (*) and systole (#) or with the exact P value.

Fig. 3.

Flow velocity (FV) is increased after exposure to a nitric oxide donor. A: representative instantaneous FV profiles for single erythrocytes in the dorsal aorta of control and S-nitroso-l-glutathione (GSNO)-treated zebrafish larvae. Index time 0 (dotted line) denotes the onset of systole. B: average maximum FV in control and GSNO-treated zebrafish larvae. Data are expressed as means ± SE for n = 9 measurements per group. *P = 0.0195.

Fig. 4.

Measurement of ventricular size and function with modulation of nitric oxide signaling. A: ventricular area in control, GSNO-treated, and 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ)-treated embryos. B: short-axis ventricular diameter measured in control and treated embryos. Statistically significant differences are denoted with actual P value when a significant difference was measured. P values above the bars refer to differences in end-diastolic measurements, while those below reference end-systolic data. n.s., Not significant. C and D: measurement of ventricular contractility by 2-dimensional FAC (C) and FS (D). Data are expressed as means ± SE for n ≥ 9 measurements per group. In A and B, end-diastolic measurements are shown by open bars and end-systolic measurements are shown by filled bars; significant differences are denoted with exact P value. In C, statistically significant differences are denoted by * (P = 0.0074 compared with control), ** (P < 0.0001 compared with control and GSNO), or n.s. (no significant difference was measured). In D, * denotes P = 0.0022 compared with control and ** denotes P < 0.0001 compared with both control and GSNO-exposed embryos.

Fig. 5.

Drug treatment modulates cardiovascular function in developing zebrafish. A: SAx diameter at both end systole (ES; filled bars) and end diastole (ED; open bars) after drug treatment. Significant differences at ES are noted below the bars, while significant ED differences are identified above the open bars. B: FS following drug treatment; significant differences are as noted. C: dorsal aortic peak FV following isoproterenol treatment. Statistically significant differences are presented with exact P values in A and B; in C * denotes P < 0.05.

Fig. 6.

Drug treatment confirms increased ventricular performance with β-adrenergic stimulation. A: SAx diameter at both ES and ED after treatment with epinephrine and dobutamine. Epinephrine and dobutamine treatment (5 μg/ml) cause a decrease in SAx diameter at ES but do not impact the ED dimension. B: calculation of FS shows augmented contractility with drug treatment. C: measurement of myocardial thickness demonstrates increased ES but not ED wall thickness with β-adrenergic agonist exposure. *P < 0.05; n.s., nonsignificant difference.