Abstract
Zebrafish embryos and larvae have emerged as an excellent model in cardiovascular research and are amenable to live imaging with genetically encoded biosensors to study cardiac cell behaviours, including calcium dynamics. To monitor calcium ion levels in three to five days post-fertilization larvae, we have used bioluminescence. We generated a transgenic line expressing GFP-aequorin in the heart, Tg(myl7:GA), and optimized a reconstitution protocol to boost aequorin luminescence. The analogue diacetyl h-coelenterazine enhanced light output and signal-to-noise ratio. With this cardioluminescence model, we imaged the time-averaged calcium levels and beat-to-beat calcium oscillations continuously for hours. As a proof-of-concept of the transgenic line, changes in ventricular calcium levels were observed by Bay K8644, an L-type calcium channel activator and with the blocker nifedipine. The β-adrenergic blocker propranolol decreased calcium levels, heart rate, stroke volume, and cardiac output, suggesting that larvae have a basal adrenergic tone. Zebrafish larvae treated with terfenadine for 24 h have been proposed as a model of heart failure. Tg(myl7:GA) larvae treated with terfenadine showed bradycardia, 2:1 atrioventricular block, decreased time-averaged ventricular calcium levels but increased calcium transient amplitude, and reduced cardiac output. As alterations of calcium signalling are involved in the pathogenesis of heart failure and arrhythmia, the GFP-aequorin transgenic line provides a powerful platform for understanding calcium dynamics.
Highlights
Introduction iationsCardiovascular diseases are the leading cause of death worldwide and are among the most challenging to diagnose and treat due to the complexity of their pathophysiology.Ca2+ plays a pivotal role in the excitation–contraction coupling of the heart and alterations in Ca2+ cycling and its associated proteins and pathways may trigger pathological disorders [1,2]
Tg(myl7:GCaMP)s878 adult zebrafish were outcrossed to wild-type strain and fertilized eggs at 1-cell stage were injected with 2 ng of the morpholino oligomer tnnt2a
Upon Ca2+ binding to aequorin, there is energy transfer between the excited state of CTZ and the GFP so that light emission is shifted to the green (Figure 1A) [27,33,38]
Summary
Fish used in this study were housed under standard conditions as previously described [32]. The Ca2+ biosensor GFP-aequorin (GA) [33] was cloned into the pT2A-Tol2-myl7MCS transposon vector (0.8 kb myl promoter), using XhoI and EcoRI restriction sites. To generate stable transgenic zebrafish, tol2-myl7:GA plasmid (12.5 ng/μL) was co-injected with transposase mRNA (12.5 ng/μL) in fertilized eggs of Tübingen zebrafish. Injected embryos were screened by fluorescence for GA expression in the heart and grown to adulthood (F0). The adult F0 generation was outcrossed to wild-type zebrafish to identify founders with insertions in the germline. F2 Tg(myl7:GA) heterozygous larvae were used throughout the study
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