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Improving Diagnosis and Treatment of Catecholaminergic Polymorphic Ventricular Tachycardia: Integrating Clinical and Basic Science

Subject Area Human Genetics
Cardiology, Angiology
Term from 2016 to 2019
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 287718088
 
Final Report Year 2020

Final Report Abstract

The main aim of the project was to enhance the functional understanding of ryanodine receptor 2 (RYR2) mutations in CPVT by using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). To this end, four objectives were addressed: (1) Generation and characterization of iPSCs from CPVT patients with different RYR2 mutations; (2) Investigation of electrophysiological phenotypes of CPVT iPSC-CMs; (3) Determination of the effects of different RYR2 mutations on Ca2+ handling properties in iPSC-CMs; and (4) Understanding the function of RYR2 during CM differentiation. We generated and characterized iPSCs from CPVT patients with different RYR2 mutations (G357S, R420Q, R420W, E1724K, A2254V, E4076K, F4739Lfs*15, and H4742Y) by using the RNA Sendai virus system. To investigate the phenotypes of iPSC-CMs, we first established a simple and efficient method for differentiation of iPSCs into defined functional CM subtypes and demonstrated that atrial and ventricular iPSC-CMs highly correspond to the atrial and ventricular heart muscle, respectively, supporting their suitability in disease modelling. To overcome the high workload and low output for electrophysiological analyses using the manual patch clamp technology, we established an automated patch clamp technology for studying ionic currents and action potential in iPSC-CMs. By applying the established methods, we showed that CPVT iPSC-CMs could recapitulate the disease phenotypes (stress-dependent arrhythmia) but revealed mutation-specific differences in electrophysiological properties. We then deeply investigated the calcium handling, including Ca2+ sparks, store-overload induced Ca2 release, calcium and caffeine sensitivity of RYR2, and the L-type calcium channels. In summary, the A2254V variation presented a typical gain-of-function mutation, rendering the RYR2 hyperactivity, while E4076K was identified as a loss-of-function mutation, leading to hypoactive RYR2. R420W and H4742Y mutations did not enhance or suppress the activity of RYR2. However, by destabilizing the N-terminal domain of RYR2, R420W caused Ca2+ leak, which could be blocked by RYR2 inhibitor. The H4742Y mutation led to a consistent and inhibitor-resistant Ca2+ leak via RYR2, suggesting a structural remodeling of RYR2 that disturbs complete closure of the channel. These results confirmed the importance of RYR2 in the maintenance of Ca2+ handling and gained evidences that the underlying mechanisms of CPVT caused by different mutations in RYR2 should be mutation-specific rather than unified. These explain why cellular phenotypes of iPSC-CMs with these mutations are different. Further studies are necessary to figure out whether other ion channels (for example, NCX) are involved in the physiological abnormality of CPVT iPSC-CMs. Furthermore, by applying the CRISPR-cas9 technology in iPSCs, we modified the CPVT iPSC line with the mutation E4076K and generated one cell line (T42), which has a healthy allele and an allele with a premature termination codon (E4074Gfs*24). We found that T42-iPSC-CMs exhibit normal action potential at Iso-challenged conditions whereas their parental CPVT iPSC-CMs reveal stress-dependent arrhythmia. Moreover, T42-iPSC-CMs showed significantly lower SR calcium leak compared to CPVT iPSC-CMs, but comparable to Ctrl-CMs. These results suggest that the dysfunction of iPSC-CMs with a RYR2 mutation can be restored by the deletion of the allele with the mutation, suggesting gene editing-based strategies as a potential treatment option for CPVT. Furthermore, we have generated 3 homozygous RYR2 knockout human iPSC lines (X4, A3 and A5). Our data demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function. IP3R-mediated Ca2+ release is the major compensatory mechanism for Ca2+ cycling in human CMs with the RYR2 deficiency.

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