Background – Zebrafish (Danio rerio), a well-established model of human cardiac disease [1], has the  ability to regenerate rapidly after injury [2].  Cardiac recovery after injury is particularly rapid in the larval phase [3]. As zebrafish possesses a similar ventricular action potential and ion channel protein expression to human heart [4], we explored the possibility of using non-invasive electrical recording in the larval stage to measure changes in cardiac electrophysiology along with gene expression during regeneration.

Methods – A PALM MicroBeam laser (Zeiss) was used to produce precise ventricular damage in n=6 transgenic larvae [Tg(myl7:GFP)] at 72 hours post-fertilisation anaesthetized in tricaine (160 mg/L).   Cardiac recovery was monitored under tricaine anaesthesia using in vivo electrical extracellular recording at 2 and 24 hours after injury.  Cryo-injury, using a cooled copper probe, was used to induce injury in n=6 anaesthetized adult zebrafish hearts, n=6 adults were used as sham. Quantitative PCR (Q-PCR) was used to measure gene expression. For statistics Student’s t-tests for paired and unpaired data were used.

Results – Larvae showed regular sequences of wave-forms (a small atrial followed by a larger ventricular depolarization). Larvae (n=6) at 2h post-injury showed one of three states of electrical activity (chaotic small amplitude waves, an atrial depolarisation and no ventricular depolarisation, or an atrial depolarisation and a reduced amplitude ventricular depolarisation).  Remarkably, recordings from the same larvae 24h post-injury showed recovery of the normal sequence and amplitude of depolarization (p<0.05). Q-PCR analysis in adult hearts showed significant increases in the expression of key ion channel genes in zebrafish at 3 and 7 days post-cryoinjury compared to sham.  These genes significantly altered were Scn5lab (ortholog of the human cardiac Na⁺ channel;  p<0.001) [5],  KcnK1b (a K⁺ two-pore  domain K+ channel, expressed in  heart that acts upstream of or within heart process;  p<0.0001),  Cacna1ab (a voltage-gated calcium channel subunit alpha Cav2.1; p<0.01)  and  regulatory genes  RelA and  Tmem161 (highly conserved regulator of cardiac rhythm that function to modulate ion channel activity in zebrafish; p<0.001) [6].

Conclusions – We characterised, for the first time, the changes of the electric phenotype in zebrafish larval hearts following injury. This electrical phenotype is highly reproducible and could be mediated by changes in the expression of membrane ion channel genes, as it was observed in the adult injured zebrafish heart.

Ethical standards – Zebrafish used in this study were maintained in the zebrafish facility of the University of Edinburgh according to standard procedures and ethical guidelines.