Induced pluripotent stem cell-derived cardiomyocytes as model for studying CPVT caused by mutations in RYR2

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a severe hereditary cardiac ion-channel disease, which is caused by mutations in ryanodine receptor 2 (RYR2) or calsequestrin 2 (CASQ2). After β-adrenergic stimulation, these mutations lead to unwanted Ca2+ release from the sarcoplasmic reticulum (SR), which can cause life threatening arrhythmias, and sudden cardiac death. However, the underlying mechanisms how exactly point mutations in RYR2 induce arrhythmias have not been completely understood. Aim of this project was to establish in vitro disease models using human induced pluripotent stem cells (hiPSCs) as unlimited source for cardiomyocytes (CMs) and to study the molecular mechanism of CPVT. In this study, hiPSCs were generated from three patients with CPVT symptoms caused by the heterozygous point mutations, A2254V and E4076K, in the gene RYR2. The generated hiPSCs showed pluripotency and were able to differentiate spontaneously into derivatives of all three germ layers. CPVT-hiPSCs and Ctrl-hiPSCs from healthy subjects were directly differentiated into functional ventricular CMs and tested for their purity by their expression of cardiac-specific marker proteins. Allele-specific sequencing showed balanced allelic expression of the disease-causing allele and healthy allele of RYR2 in CPVT-CMs. Western blot analysis showed that CPVT-CMs expressed similar levels of RYR2 in comparison to Ctrl-CMs. RYR2 phosphorylation (Ser2808, Ser2814) was not altered in CPVT-CMs carrying mutations R420W, A2254V and E4076K at basal or isoprenaline-stimulated conditions when compared to Ctrl-CMs. This result refutes the hypothesis that stress induces hyperphosphorylation of RYR2 due to missense mutations in the RYR2 gene, at least not in CPVT-CMs with the mutations analyzed. Furthermore, the CRISPR/Cas9 system was utilized for genome editing in hiPSCs. Homogeneous as well as heterogeneous CRISPR/Cas9-edited hiPSCs were generated and differentiated into CMs. CMs derived from RYR2+/O-T42-hiPSCs, which contain a heterogeneous deletion of 17 bps in the RYR2 gene leading to a premature termination codon, manifested downregulated mRNA expression of the CPVT-causing allele. However, RYR2 expression in RYR2+/O-T42-CMs was not altered and comparable to CPVT-CMs. Moreover, RYR2+/O-T42-CMs showed significantly reduced Ca2+ sparks, which were comparable to Ctrl-CMs, suggesting a potential healthy phenotype of CRISPR/Cas9-edited RYR2+/O-T42-CMs. In contrast, CRISPR/Cas9-edited CMs with a homozygous deletion in RYR2 (RYR2O/O-A3-CMs) showed normal RYR2 mRNA expression but no expression of the RYR2 protein. Interestingly, RYR2O/O-A3-CMs showed nearly no SR Ca2+ leak due to the missing RYR2 protein. Taken together, the data demonstrate that patient-specific hiPSCs can be used to model CPVT. In CPVT-CMs with the mutations analyzed, the Ca2+ leak is not due to stress-induced hyperphosphorylation of RYR2. Knockdown of the CPVT-causing RYR2 allele can rescue, at least partially, the disease phenotype in CPVT-CMs. In addition, the data suggest that RYR2 is not required for the initiation of differentiation from hiPSCs into CMs.