Functional Assessment of Cardiac Responses of Adult Zebrafish (Danio rerio) to Acute and Chronic Temperature Change Using High-Resolution Echocardiography

Abstract

The zebrafish (Danio rerio) is an important organism as a model for understanding vertebrate cardiovascular development. However, little is known about adult ZF cardiac function and how contractile function changes to cope with fluctuations in ambient temperature. The goals of this study were to: 1) determine if high resolution echocardiography (HRE) in the presence of reduced cardiodepressant anesthetics could be used to accurately investigate the structural and functional properties of the ZF heart and 2) if the effect of ambient temperature changes both acutely and chronically could be determined non-invasively using HRE in vivo. Heart rate (HR) appears to be the critical factor in modifying cardiac output (CO) with ambient temperature fluctuation as it increases from 78 ± 5.9 bpm at 18°C to 162 ± 9.7 bpm at 28°C regardless of acclimation state (cold acclimated CA- 18°C; warm acclimated WA- 28°C). Stroke volume (SV) is highest when the ambient temperature matches the acclimation temperature, though this difference did not constitute a significant effect (CA 1.17 ± 0.15 μL at 18°C vs 1.06 ± 0.14 μl at 28°C; WA 1.10 ± 0.13 μL at 18°C vs 1.12 ± 0.12 μl at 28°C). The isovolumetric contraction time (IVCT) was significantly shorter in CA fish at 18°C. The CA group showed improved systolic function at 18°C in comparison to the WA group with significant increases in both ejection fraction and fractional shortening and decreases in IVCT. The decreased early peak (E) velocity and early peak velocity / atrial peak velocity (E/A) ratio in the CA group are likely associated with increased reliance on atrial contraction for ventricular filling.

Fig 1. Longitudinal position of zebrafish.

The ultrasound transducer beam (70 MHz) was positioned at about 85 degrees to ventral surface of the anesthetized zebrafish through all protocols. ZF were placed in a 45 ml custom-made water-jacketed glass chamber with the thermal probe (shown with the blue wire) inserted into the bath in proximity to the ZF.

Fig 2. B-mode image of a long-axis view of a WA ZF heart at 28°C.

The perimeter of the ventricle represented by (A) red trace for diastole and (B) cyan trace for systole. V Area, ventricular area; V, volume; Ld, greatest length of the spline during diastole; Ls, greatest length of the splice during systole; SV, stroke volume determined by diastolic volume-systolic volume; EF, ejection fraction determined by SV/diastolic volume; FS, fractional shortening determined by (Ld—Ls) / Ld * 100.

Fig 3. Pulse wave Doppler image of ventricular inflow and outflow velocity.

Ventricular inflow image was used to measure IVRT, IVCT, ET, early (e) filling peak velocity and atrial (a) filling peak velocity in a WA ZF at: (A) 28°C and (B) 18°C. Note the difference in x-axis in the 28°C condition and 18°C condition. (C) Ventricular outflow peak velocity (VOPV) of a WA ZF at 28°C. The Y-axis indicates velocity (mm/s) and the x-axis indicates time (ms). ET, ejection time; IVCT, isovolumetric contraction time; IVRT, Isovolumetric relaxation time; Vel, velocity.

Fig 4. Ventricular functional parameters were calculated from B-mode long-axis plane (n = 10).

(A) Heart rate, (B) normalized cardiac output, (C) ejection fraction and (D) fractional shortening of both WA and CA zebrafish at two acute temperatures. Error bars represent SEM. * represents significant difference of both groups between temperatures. † represents significant interacting effect of acclimation state. Red: warm acclimated (WA) zebrafish. Blue: cold acclimated (CA) zebrafish.

Fig 5. Parameters were calculated from pulse wave Doppler mode of ventricular inflow view (n = 10).

(A) Early (e) filling peak velocity, (B) atrial (a) flow peak velocity, (C) E/A ratio and (D) isovolumic contraction time (IVCT) of CA and WA zebrafish at two acute temperatures. The ratio of early (e) filling peak velocity and atrial (a) late filling peak velocity allows for evaluation of zebrafish ventricular function. Error bars represent SEM. * represents significant difference of both groups between temperatures. † represents significant interacting effect of acclimation state. Red: warm acclimated (WA) zebrafish. Blue: cold acclimated (CA) zebrafish.