Introduction

With a prevalence of 1% of all births, congenital heart disease represents the number one death-threat to children younger than a year. Therefore, it embodies a serious challenge for modern medicine. Recently, a gene missense variant (c.1774G>A; pG592R) was identified in PRKD1, encoding protein kinase D1 (PRKD1), thus providing the first published data linking PRKD1 gene defects to congenital heart disease.

The ubiquitously expressed serine threonine kinase PRKD1 plays a crucial role in the cardiovascular system. It contributes to embryonic heart development and pathological hypertrophy in the adult heart by regulating myocyte enhancer factor 2 (MEF2)-dependent gene transcription via phosphorylation of histone deacetylases.

Aim of the study: To investigate the molecular disease mechanisms evoked by the G592R PRKD1 variant in human induced pluripotent stem cell (hiPSC)-derived cardiac organoids.

Methods and results: To introduce the patients’ variant into a wildtype hiPSC line, a CRISPR/Cas9 strategy was developed. HiPSCs were transfected with a sgRNA and the ribonucleoprotein complex by electroporation and incubated under cold-shock conditions (5% O₂, 5% CO₂, 32°C) for 48 hours. Clones were picked and presorted by introduction of a XbaI restriction site. Introduction of the variant was subsequently verified by Sanger sequencing. Four clones revealed proper introduction of the patients’ variant, thereby, two carrying the variant in the heterozygous state and two in the homozygous form.

For quality control, the ten most likely off-target loci were checked by PCR for genomic integrity. The clones showed stage-specific embryonic antigen-3 (SSEA-3) positivity of over 90%. The karyotype integrity was verified by NanoString ® analysis.

Wild-type, heterozygous and homozygous hiPSC lines were successfully differentiated to beating cardiac organoids using a 15-day differentiation protocol in a 96-well format. To investigate phenotypical differences between genotypes, cardiac organoids were characterised for their size, structure, cellular composition and morphology by FACS, immunofluorescence, electron microscopy and histology. Additionally, 3D reconstructions of the organoids were generated.

Conclusion

The patients’ variant pG592R was successfully CRISPRed in hiPSCs in heterozygous and homozygous form. The hiPSC lines were propagated and quality controlled to a high standard. From these cell lines, cardiac organoids and EHTs were successfully differentiated and are currently being characterized for putative disease-causing differences.

Outlook

Recently, our group generated engineered heart tissue (EHT) from hiPSC-derived cardiomyocytes carrying PRKD1 G592R in heterozygous (het) or homozygous (hom) form. Assessment of contractile properties revealed significantly higher force development in het and hom EHTs, accompanied by impaired contraction kinetics, compared to WT.