Simultaneous imaging of calcium and contraction in the beating heart of zebrafish larvae

Salgado-Almario, Jussep; Vicente, Manuel; Molina, Yillcer; Martinez-Sielva, Antonio; Vincent, Pierre; Domingo, Beatriz; Llopis, Juan

doi:10.7150/thno.64734

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Theranostics 2022; 12(3):1012-1029. doi:10.7150/thno.64734 This issue Cite

Research Paper

Simultaneous imaging of calcium and contraction in the beating heart of zebrafish larvae

Jussep Salgado-Almario¹, Manuel Vicente¹, Yillcer Molina¹, Antonio Martinez-Sielva¹, Pierre Vincent^2✉, Beatriz Domingo^1✉, Juan Llopis^1✉

1. Physiology and Cell Dynamics, Centro Regional de Investigaciones Biomédicas (CRIB) and Facultad de Medicina de Albacete, Universidad de Castilla-La Mancha, C/Almansa 14, 02006 Albacete, Spain.
2. Sorbonne Université, CNRS, Biological Adaptation and Ageing, UMR 8256, F-75005 Paris, France.

Citation:

Salgado-Almario J, Vicente M, Molina Y, Martinez-Sielva A, Vincent P, Domingo B, Llopis J. Simultaneous imaging of calcium and contraction in the beating heart of zebrafish larvae. Theranostics 2022; 12(3):1012-1029. doi:10.7150/thno.64734. https://www.thno.org/v12p1012.htm

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Abstract

In vivo models of cardiac function maintain the complex relationship of cardiomyocytes with other heart cells, as well as the paracrine and mechanoelectrical feedback mechanisms. We aimed at imaging calcium transients simultaneously with heart contraction in zebrafish larvae.

Methods: To image calcium in beating hearts, we generated a zebrafish transgenic line expressing the FRET-based ratiometric biosensor Twitch-4. Since emission ratioing canceled out the motion artifacts, we did not use myosin inhibitors or tnnt2a morpholinos to uncouple contraction from changes in calcium levels. We wrote an analysis program to automatically calculate kinetic parameters of the calcium transients. In addition, the ventricular diameter was determined in the fluorescence images providing a real-time measurement of contraction correlated with calcium.

Results: Expression of Twitch-4 did not affect the force of contraction, the size of the heart nor the heart rate in 3- and 5-days post-fertilization (dpf) larvae. Comparison of 3 and 5 dpf larvae showed that calcium levels and transient amplitude were larger at 5 dpf, but the fractional shortening did not change. To validate the model, we evaluated the effect of drugs with known effects on cardiomyocytes. Calcium levels and the force of contraction decreased by the L-type calcium channel blocker nifedipine, whereas they increased with the activator Bay-K 8644. Caffeine induced bradycardia, markedly decreased ventricular diastolic calcium levels, increased the size of the calcium transients, and caused an escape rhythm in some larvae.

Conclusions: The Tg(myl7:Twitch-4) line provides a physiological approach to image systolic and diastolic calcium levels in the heart of zebrafish larvae. Since the heart is beating, calcium levels and contraction can be correlated. This line will be a useful tool to address pathophysiological mechanisms in diseases like heart failure and arrhythmia, in cardiotoxicity studies and for drug screening.

Keywords: imaging, calcium, contraction, heart, zebrafish

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APA

Salgado-Almario, J., Vicente, M., Molina, Y., Martinez-Sielva, A., Vincent, P., Domingo, B., Llopis, J. (2022). Simultaneous imaging of calcium and contraction in the beating heart of zebrafish larvae. Theranostics, 12(3), 1012-1029. https://doi.org/10.7150/thno.64734.

ACS

Salgado-Almario, J.; Vicente, M.; Molina, Y.; Martinez-Sielva, A.; Vincent, P.; Domingo, B.; Llopis, J. Simultaneous imaging of calcium and contraction in the beating heart of zebrafish larvae. Theranostics 2022, 12 (3), 1012-1029. DOI: 10.7150/thno.64734.

NLM

CSE

Salgado-Almario J, Vicente M, Molina Y, Martinez-Sielva A, Vincent P, Domingo B, Llopis J. 2022. Simultaneous imaging of calcium and contraction in the beating heart of zebrafish larvae. Theranostics. 12(3):1012-1029.

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