Verbesserung der Diagnose und Behandlung von katecholaminerger polymorpher ventrikulärer Tachykardie: Einbindung von klinischer Forschung und Grundlagenforschung
Kardiologie, Angiologie
Zusammenfassung der Projektergebnisse
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.
Projektbezogene Publikationen (Auswahl)
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Making human cardiomyocytes up to date: Derivation, maturation state and perspectives. (2017) Int J Cardiol, 241:379-386
Kolanowski T, Antos C, Guan K
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Severe DCM phenotype of patient harboring RBM20 mutation S635A can be modeled by patient-specific induced pluripotent stem cell-derived cardiomyocytes. (2017) J Mol Cell Cardiol, 113:9-21
Streckfuß-Bömeke K, Tiburcy M, Fomin A, Luo X, Li W, Fischer C, Özcelik C, Perrot A, Sossalla S, Haas J, Vidal RO, Rebs S, Khadjeh, S, Meder B, Bonn S, Linke WA, Zimmermann W-H, Hasenfuß G, Guan K
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(2018) Deep phenotyping of human induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes. JCI Insight, 3:e99941
Cyganek L, Tiburcy M, Sekeres K, Gerstenberg K, Bohnenberger H, Lenz C, Henze S, Stauske M, Salinas G, Zimmermann W-H, Hasenfuss G, Guan K. Deep phenotyping of human induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes
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MicroRNAs in cardiomyocyte differentiation and maturation. (2018) Cardiovasc Res, 114:779-781
Alfar EA, El-Armouche A, Guan K
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Sarcoplasmic reticulum calcium leak contributes to arrhythmia but not to heart failure progression. (2018) Sci Transl Med, 10:eaan0724
Mohamed BA, Hartmann N, Tirilomis P, Sekeres K, Li W, Neef S, Richter C, Zeisberg EM, Kattner L, Didié M, Guan K, Schmitto JD, Lehnart SE, Luther S, Voigt N, Seidler T, Sossalla S, Hasenfuss G, Toischer K
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Establishment of an automated patch-clamp platform for electrophysiological and pharmacological evaluation of hiPSC-CMs. (2019) Stem Cell Res, 41:101662
Li W, Luo X, Ulbricht Y, Wagner M, Piorkowski C, El-Armouche A, Guan K
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Genetic variation in GNB5 causes bradycardia by augmenting the cholinergic response via increased acetylcholine-activated potassium current (I K,ACh). (2019) Dis Model Mech, 12(7). pii: dmm037994
Veerman CC, Mengarelli I, Koopman CD, Wilders R, van Amersfoorth SC, Bakker D, Wolswinkel R, Hababa M, de Boer TP, Guan K, Milnes J, Lodder EM, Bakkers J, Verkerk AO, Bezzina CR
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Electrophysiological Abnormalities in VLCAD Deficient hiPSC-Cardiomyocytes can be Improved by Lowering Accumulation of Fatty Acid Oxidation Intermediates (2020) Int J Mol Sci, 21(7):2589. pii: E5105
Knottnerus SJG, Mengarelli I, Wüst RCI, Baartscheer A, Bleeker JC, Coronel R, Ferdinandusse S, Guan K, IJlst L, Li W, Luo X, Portero VM, Ulbricht Y, Visser G, Wanders RJA, Wijburg FA, Verkerk AO, Houtkooper RH, Bezzina CR
