In Vivo Imaging of Zebrafish Larvae Heart Regeneration

Summary

This article presents a protocol for in vivo imaging combining confocal and lightsheet microscopy to study cardiac regeneration dynamics using the Tg(vmhc:mCherry-NTR) zebrafish line for chemogenetic targeted cardiomyocyte ablation. This enables rapid, high-fidelity longitudinal monitoring of myocardial repair.

Abstract

Cardiovascular disease is the leading cause of death globally, so learning about regeneration in the heart is a critical research topic. In the past, cardiomyocytes were thought to be terminally differentiated, and thus, the heart as an organ was thought to lack significant repair capacity. But newer data contradict it, depicting the MI and repair processes in a more active manner. Although histopathology can provide crucial structural information, it is limited to static snapshots of tissue structure; in vivo imaging, on the other hand, allows direct follow-up of regeneration kinetics. Considering the robust regenerative ability of zebrafish, they represent a good model for studying cardiac repair. Herein, this study describes an experimental protocol to visualize myocardial injury and repair in transgenic zebrafish larvae. We applied confocal microscopy and light-sheet microscopy to observe the processes of cardiomyocyte ablation and regeneration. It is an easier way to visualize in vivo, allowing real-time observation of high-resolution spatiotemporal processes of cardiac repair.

Introduction

Zebrafish embryos, due to their optical clarity, have been established as an excellent system for understanding the mechanisms of cardiac regeneration^1,2,3,4,5,6. Starting at 24 hours post-fertilization (hpf), zebrafish embryos begin beating their hearts; cardiac looping is complete at 36 hpf, and mature function occurs at 96 hpf⁷, making it an attractive model for studying early regeneration processes^8,

Protocol

All animal experiments are performed strictly according to the guidelines approved by the Animal Ethics Committee of Fujian Maternity and Child Health Hospital (No. AEC SFY 2025 058). The Tg(vmhc:mCherry-NTR) strain is kindly provided by Prof. Ruilin Zhang (Wuhan University). The Tg(myl7:EGFP) strain is obtained from the China Zebrafish Resource Center (Wuhan, China). Adult zebrafish are kept on a 14:10 h light/dark cycle in a recirculating aquaculture system at 28 °C¹⁵.

1. Zebrafish maintenance and larvae preparation: 0-96 hpf

1. Breeding and husbandry
   1. Mai....

Results

The experimental procedure is depicted in Figure 1. Following MTZ treatment, ventricular cardiomyocytes displayed acute injury on 1 dpt, marked by significant cell edema, diminished mCherry fluorescence, and a corresponding decrease in ventricular surface area. The injury peaked on 2 dpt, with edematous cells partially filling the ventricular lumen. However, by 3 dpt, clear signs of regeneration and recovery emerged, evidenced by reduced edema and the removal of cellular debris. By 4 dpt, th.......

Discussion

Considering the dynamic nature of the process of myocardial injury and regeneration, in vivo imaging has been an indispensable instrument for understanding these processes. Due to advances in gene-editing and optical engineering, this visualization method offers a significant advantage over conventional histopathology¹⁷. Although histological analysis is still useful for revealing structural information about tissue/cell, it can only provide a static cross-sectional image and requires tis.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 mm cell culture Dish	BDBIO	H802004	Drug exposure in zebrafish
1M Tris-HCl, pH8.8	BEYOTIME	ST788	Adjust the pH value of the anesthetic agent
Confocal laser scanning microscopy	Leica	TCS SP8	Observation sample
Fluorinated propylene heat shrink tube	Zeus	AS 23053/11 (Custom-made, inner diameter 1.7 mm, wall thickness 0.05 mm)	Light-sheet in vivo imaging for zebrafish
Imaris software	Imaris software	v9.9.1	3D Visualization
Light-sheet microscope	Bruker	MuVi-SPIM, Luxendo Light-Sheet,	Observation sample
Low-melting-point agarose	Invitrogen	16520-100	Fixed sample
Metronidazole	Merck	M3761	Myocardial ablation drugs
N-phenylthiourea	Merck	P7629	Eliminate melanin
Stereomicroscope	Nikon	SMZ745T	Observation sample
Tricaine	Merck	E10521	Anesthesia sample