https://doi.org/10.1007/s00392-024-02526-y
1Universitäres Herz- und Gefäßzentrum Hamburg Klinik für Kardiologie mit Schwerpunkt Elektrophysiologie Hamburg, Deutschland; 2Universitätsklinikum Hamburg-Eppendorf Institut für Experimentelle Pharmakologie und Toxikologie Hamburg, Deutschland; 3Universitätsklinikum Hamburg-Eppendorf Institut für Klinische Pharmakologie und Toxikologie Hamburg, Deutschland; 4University of Milan Humanitas Clinical and Research Center Rozzano (Milan), Italien
Background:
The project originated from the clinical observations of a left ventricular non-compaction and sudden cardiac death overlap syndrome in a large family in northern Italy. Whole exome sequencing of family members across different generations identified a single nucleotide polymorphism in the RYR2 gene (RYR2 c.5654G>A homozygous – RyR2 p.G1885E - exon 37: missense) inherited in an autosomal-recessive manner. This mutation is located in the DR3 region of the RyR2 protein, near the FKBP12.6 binding site.
Methods and Results:
Human induced pluripotent stem cells (hiPSCs) were generated from patient-derived peripheral blood mononuclear cells using a non-integrative Sendai virus protocol, with cells carrying the variant in both homozygous and heterozygous forms. These patient-derived hiPSCs successfully differentiated into cardiomyocytes using a monolayer protocol. Subsequently, 3D strip-format, force-generating Engineered Heart Tissues (EHTs) were cast for 3D structural and functional characterization. Over 60 days, the EHTs with the homozygous pathogenic variant demonstrated a progressive reduction in both beating rate and contractility, increased spontaneous arrhythmogenicity, and reduced inotropic response to isoprenaline, compared to heterozygous cells and unrelated wild-type controls. Starting from day 30 post-casting, the patient-derived EHTs exhibited significant morphological changes, including marked cellular overgrowth and reduced troponin expression. Transcriptomic analysis at 30 and 60 days post-EHT generation complemented the phenotyping and provided molecular insights into the underlying pathophysiology. To confirm the pathogenicity of the genetic variant, homozygous patient-derived hiPSCs were corrected to both homozygous and heterozygous states using CRISPR/Cas9 technology. The genetically corrected 3D tissue models displayed a reversal of the observed phenotype, showing normal beating rates, no reduction in force generation over time, and absence of spontaneous arrhythmogenicity.
Conclusion:
We identified a novel recessive RYR2 variant responsible for a familial left ventricular non-compaction and sudden cardiac death overlap syndrome. Using 3D cardiac tissue modeling with patient-derived and gene-edited hiPSCs, we uncovered a proarrhythmic and structural phenotype specifically associated with this variant. This discovery enhances our understanding of RYR2 receptor biology, illuminating a protein region previously considered to function merely as an adaptor site.