Introduction: Next-generation sequencing has generated many gene variants associated with sudden cardiac death in diseases like cardiac conduction disease (CCD). Determining which of these are causative for human disease is a major challenge. Zebrafish have emerged as an attractive alternative for modeling human heart disease due to similarities in its physiology, together with ease of genetic manipulation, external development, and optical transparency.
Methods and Results: We performed whole-exome DNA sequencing for 2 patients with early-onset CCD and found LMNA protein truncating variants c.339 dup T and c.1489-2A>G, respectively. Both patients underwent a pacemaker implantation at the ages of 42 and 23, respectively, and had a family history of pacemaker implantation and sudden cardiac death. We sought to generate and characterize a knockout zebrafish with CRISPR-mediated insertions or deletions (indels) of the human LMNA homolog, lmna. We microinjected a single-guide RNA and Cas9 protein into 1 cell stage zebrafish embryos and prepared genomic DNA from these CRISPR/Cas9 injected embryos. As such, we confirmed a variety of indels at the site of double strands break in the lmna gene by Sanger sequencing. Then, we evaluated cardiac physiology of these embryos at 48 hpf, and performed voltage mapping on isolated 72 hpf zebrafish hearts. At 48hpf, the mean heart rate (bpm) of the CRISPR/Cas9 injected embryos with lmna indels was significantly decreased compared to that of the CRISPR only injected embryos (c.339 dup T, 127 ± 10 vs 136 ± 8; c.1489-2A>G, 131 ± 14 vs 142 ± 14; p<0.05, respectively). In addition, optical mapping studies demonstrated mean conduction velocities (mm/sec) were significantly decreased in the CRISPR/CAS9 injected embryos compared to control embryos (c.339 dup T, 8.0 ± 1.2 vs 16.8 ± 2.6; c.1489-2A>G, 6.7 ± 0.8 vs 9.9 ± 1.1; p<0.05, respectively). The evaluation of cardiac function also showed that both the ejection fraction and cardiac output of CRISPR/Cas9 injected embryos were significantly decreased compared to control embryos.
Conclusions: CRISPR-mediated indels of the human LMNA homolog in zebrafish recapitulated the cardiac phenotypes of early-onset CCD.