Quantitative Analysis of Calcium Dynamics in Beating Larval Zebrafish Heart

Yu-Cheng Chuang^1*, Praveen Kumar¹, Ian Liau¹

¹Institute of Molecular Science and Department of Applied Chemistry, National Chiao Tung University, Hsinchu City, Taiwan

* Presenter:Yu-Cheng Chuang, email:andy.yc.chuang@gmail.com

Calcium flow regulates not only the contraction of single cardiac myocytes but also the rhythm of heartbeat. While determination of intracellular calcium level on single cells have been demonstrated with molecule- or protein- based fluorescent indicators, imaging of calcium wave on a rapidly beating heart in vivo remains challenging. Here we report the visualization of calcium flow on living larval zebrafish (4 dpf) that express GCaMP, a fluorescent indicator protein of calcium, in cardiac myocytes. To reconstruct 3D images of a beating heart, we developed a hybrid imaging system, in which the onset of confocal image acquisition (25 to 50 fps) was synchronously triggered with the cardiac rhythm larval zebrafish (3 to 3.5 Hz) monitored optically from a side-view microscope. With such setup, we were able to acquire time-lapse 2D images synchronous to a selected phase in a cardiac cycle at varied z-depth (total of 120 μm, interval = 1 μm). These images (more than 8000 frames) were then processed off-line using our home-made computer code to reconstruct dynamic 3D images of a beating heart. To image the calcium wave, we prepared zebrafish larvae expressing both GCaMP and DsRed in cardiac myocytes, and constructed pseudodynamic 3D cardiac images based on the fluorescence of both proteins. To correct the the artifact resulting from the contraction and movement of cardiac myocytes during heartbeat, we further normalized the fluorescence intensity of GCaMP with that of DsRed. The resulting normalized GCaMP intensity of individual cardiac myocytes then represents the intracellular calcium level of individual cardiac myocytes at each time-point in a cardiac cycle. Our result delicately reveals the spatiotemporal profile of the calcium wave on a beating heart of larval zebrafish, and shows that the cardiac calcium wave in zebrafish is profoundly consistent with the cardiac conduction pathway of human heart. In summary, we developed a phase-selective, synchronously triggered imaging system and a fluorescence-image normalization method to quantitatively evaluate calcium dynamics and cardiac function of a beating larval zebrafish heart, which should open a route for not only fundamental studies of cardiac physiology but also screening of drugs with cardiac activity or toxicity.

Keywords: pseudodynamic imaging, confocal microscopy, zebrafish, calcium wave, cardiac function