In vivo assessment of cardiac morphology and function in heart-specific green fluorescent zebrafish

Background/Purpose: The zebrafish (Danio rerio) is a new animal model for cardiac research. Zebrafish possessing a green fluorescent heart facilitates the dynamic observation of cardiac development, morphology, and function in vivo. However, the effect of an excessive expression of green fluorescent protein (GFP) in cardiac muscle on the heart function of zebrafish has not been reported. Methods: We cloned a 1.6 kb polymerase chain reaction (PCR) product containing the upstream sequence (870 bp), exon 1 (39 bp), intron 1 (682 bp), and exon 2 (69 bp) of the zebrafish cardiac myosin light chain 2 gene. A germ line-transmitted zebrafish possessing a green fluorescent heart was generated by injecting this PCR product fused with the GFP gene with ends consisting of inverted terminal repeats of an adeno-associated virus. Results: Green fluorescence was intensively and specifically expressed in the myocardial cells located around both the heart chambers. Two lines with different GFP expression were bred (A26 and A277). The luminance of A277 was brighter than that of A26 (1.7-fold). The 4 days postfertilization (dpf) cardiac function and morphology were similar between these two groups. However, the 8 dpf cardiac growth seemed to be retarded in the A277 group. The 8 dpf heart rate, stroke volume, and cardiac output were also significantly lower in the A277 group. Conclusion: Excess expression of GFP seems to exert some detrimental effects on zebrafish hearts.