3D-Modelling of a RyR2-associated left ventricular non-compaction and sudden cardiac death overlap syndrome

Background: Genetic cardiomyopathies constitute a wide spectrum of inherited heart diseases. Multiple underlying genetic variants have been discovered recently. The current project arises from the discovery of a genetic variant, responsible for a left ventricular non-compaction (LVNC) and sudden cardiac death (SCD) overlap syndrome, in a large family in Northern Italy. Methods and Results: We reconstructed three-generation pedigree. The phenotypical presentation included a high incidence of SCD and variable expression of LVNC leading to advanced heart failure. We identified, through whole exome-sequencing, a novel ryanodine receptor type 2 (RYR2) variant, inherited in an autosomal recessive fashion. Induced pluripotent stem cells (hiPSCs) were generated from a proband carrying the homozygous Gly1885Glu variant and from his healthy heterozygous daughter. A chemically defined monolayer protocol, based on sequential activation and inhibition of WNT signaling, was used to efficiently obtain patient-derived cardiomyocytes (CMs). The cells were subsequently used to cast patient-derived 3D strip-format, force-generating Engineered Heart Tissues (EHTs). Contraction analysis over 60 days showed marked differences in force development of the patient-derived lines. In particular, the EHTs carrying the homozygous pathogenic variant showed progressive reduction in both beating rate and force over time, morphological changes with areas of marked cellular overgrowth and reduced troponin expression, increased arrhythmogenicity at the steady state and reduced inotropic response after exposure to isoprenaline, compared to the heterozygous line and unrelated healthy controls. Moreover, starting from the patient cell line, we generated the isogenic control line through CRISPR-Cas9 correction of the variant on both alleles. Conclusions: We describe the genotype-phenotype correlation between a novel homozygous RYR2 variant responsible for a LVNC and SCD overlap syndrome. Disease modelling with hiPSCs supports the causative role of the variant discovered in the development of arrhythmias and heart failure.