Abstract
BackgroundWhile the adult zebrafish (Danio rerio) heart demonstrates a remarkable capacity for self-renewal following apical resection little is known about the response to injury in the embryonic heart.MethodsInjury to the beating zebrafish embryo heart was induced by laser using a transgenic zebrafish expressing cardiomyocyte specific green fluorescent protein. Changes in ejection fraction (EF), heart rate (HR), and caudal vein blood flow (CVBF) assessed by video capture techniques were assessed at 2, 24 and 48 h post-laser. Change in total and mitotic ventricular cardiomyocyte number following laser injury was also assessed by counting respectively DAPI (VCt) and Phospho-histone H3 (VCm) positive nuclei in isolated hearts using confocal microscopy.ResultsLaser injury to the ventricle resulted in bradycardia and mild bleeding into the pericardium. At 2 h post-laser injury, there was a significant reduction in cardiac performance in lasered-hearts compared with controls (HR 117 ± 11 vs 167 ± 9 bpm, p ≤ 0.001; EF 14.1 ± 1.8 vs 20.1 ± 1.3%, p ≤ 0.001; CVBF 103 ± 15 vs 316 ± 13μms− 1, p ≤ 0.001, respectively). Isolated hearts showed a significant reduction in VCt at 2 h post-laser compared to controls (195 ± 15 vs 238 ± 15, p ≤ 0.05). Histology showed necrosis and apoptosis (TUNEL assay) at the site of laser injury. At 24 h post-laser cardiac performance and VCt had recovered fully to control levels. Pretreatment with the cell-cycle inhibitor, aphidicolin, significantly inhibited functional recovery of the ventricle accompanied by a significant inhibition of cardiomyocyte proliferation.ConclusionsLaser-targeted injury of the zebrafish embryonic heart is a novel and reproducible model of cardiac injury and repair suitable for pharmacological and molecular studies.
Highlights
The adult mammalian heart has little or no proliferative capacity [1,2] while the neonatal mouse heart retains the capacity to regenerate for up to 7 days following birth [3]
A single laser pulse to the mid-cavity of the ventricle (Fig. 1) resulted in instantaneous cardiac injury confirmed optically by a 2 to 4 s pause followed by marked bradycardia and a small amount of bleeding into the pericardial space
There followed a gradual and progressive increase in heart rate (HR) over the following 2 to 3 min with a significantly reduced HR compared with controls by 2 h post-laser (117 ± 11 vs 157 ± 9 bpm, p ≤ 0.001, Fig. 2B)
Summary
The adult mammalian heart has little or no proliferative capacity [1,2] while the neonatal mouse heart retains the capacity to regenerate for up to 7 days following birth [3]. The adult zebrafish heart undergoes a regenerative process following resection or cryo-injury which results in complete functional recovery [4]. The regenerative process in the adult zebrafish heart has been ascribed to dedifferentiation and proliferation of existing cardiomyocytes which demonstrate reactivation of a number of cardiac markers typically observed during early embryonic development [5]. At 2 h post-laser injury, there was a significant reduction in cardiac performance in lasered-hearts compared with controls (HR 117 ± 11 vs 167 ± 9 bpm, p ≤ 0.001; EF 14.1 ± 1.8 vs 20.1 ± 1.3%, p ≤ 0.001; CVBF 103 ± 15 vs 316 ± 13μms−1, p ≤ 0.001, respectively). Isolated hearts showed a significant reduction in VCt at 2 h post-laser compared to controls (195 ± 15 vs 238 ± 15, p ≤ 0.05). Conclusions: Laser-targeted injury of the zebrafish embryonic heart is a novel and reproducible model of cardiac injury and repair suitable for pharmacological and molecular studies
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