Modeling inherited short-coupled polymorphic ventricular tachycardia using patient specific hiPSC-derived cardiomyocytes and CRISPR/Cas 9 technology

Polymorphic ventricular tachycardia (PMVT) can occur in patients with structurally normal hearts and in 8% of cases can lead to sudden cardiac death, typically exercise-induced. The role of the cardiac type 2 ryanodine receptor (RyR2) in pathogenesis of PMVT presenting at rest is unclear. We aimed here at modelling PMVT observed in a patient harboring the RyR2-H29D mutation by comparing the molecular and functional properties of RyR2-H29D hiPSC-derived cardiomyocytes (hiPSC-CMs) with their isogenic control counterparts with a particular focus on the RyR2 properties. We collected blood samples from the patient and generated several clones of RyR2-H29D hiPSC, in addition to generating an isogenic control by reverting the RyR2-H29D mutation using CRIPSR/Cas9 technology. We used fluorescent confocal microscopy, patch-clamp and video-image-based analysis to investigate the molecular and functional consequences of the RyR2-H29D mutation. We first hypothesized that PMVT hiPSC-CMs expressing the RyR2-H29D mutation would exhibit abnormal Ca 2+ homeostasis. Thus, we measured and analyzed the intracellular Ca 2+ variation. We found that the RyR2-H29D hiPSC-CMs exhibit clone-independent aberrant properties including intracellular sarcoplasmic reticulum (SR) Ca 2+ leak through RyR2 under physiological pacing. The contribution of inositol 1,4,5-trisphosphate receptors to excitation-contraction coupling exacerbate the abnormal intracellular Ca 2+ release in the RyR2-H29D hiPSC-CMs. Moreover, the RyR2-H29D hiPSC-CMs exhibit RyR2 post-translational remodeling, shorter action potentials, delayed afterdepolarizations, arrhythmias and aberrant contractile properties compared to isogenic controls. These abnormalities are fully reversed with isogenic control. Our results suggest that RyR2-mediated Ca 2+ leak induces an impairment of Ca 2+ homeostasis and provide support to decipher the molecular mechanisms of short-coupled PMVT at rest.