Investigation of pathophysiolo­gical mechanism in induced pluripotent stem cell-derived cardiomyocytes from CPVT patients

In adult cadiomyocytes (CMs), ryanodine receptor 2 (RYR2) is an indispensable Ca²⁺ release channel that ensures the integrity of excitation-contraction (E-C) coupling, which is fundamental for every heartbeat. However, the role and importance of RYR2 during human embryonic cardiac development are still poorly understood.

In this study, after the knockout of RYR2 gene (RYR2^–/–), induced pluripotent stem cells (iPSCs) were able to differentiate into cardiomyocytes (CMs) with an efficiency similar to control iPSCs (Ctrl-iPSCs); however, the survival of iPSC-CMs was markedly affected by the lack of functional RYR2. While Ctrl-iPSC-CMs exhibited regular Ca²⁺ handling, significantly reduced frequency and intense abnormalities of Ca²⁺ transients were observed in RYR2^–/–-iPSC-CMs. These findings demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a life-threatening inherited arrhythmogenic disorder. RYR2 mutations are the genetic cause of CPVT Type 1. So far, the pathogenic mechanism of how RYR2 mutations remodel cardiac rhythm remains controversial, because all existing hypotheses cannot independently and universally represent the mechanism behind CPVT. Patient-specific iPSCs offer a unique opportunity for CPVT modeling and investigation in vitro. In this study, the effects of four different RYR2 mutations (R420W, A2254C, E4076K, and H4742Y) on cardiac Ca²⁺ handling were examined individually.

In summary, the A2254V variation presented a typical gain-of-function mutation, rendering the RYR2 hyperactive, while the E4076K variation 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. These results confirmed the importance of RYR2 on the maintenance of Ca²⁺ handling and gained evidence for the theory that the underlying mechanisms of CPVT caused by mutations in RYR2 should be mutation-specific rather than unified.

This study suggests hiPSC-CMs as a suitable platform for modeling cardiac arrhythmogenic disease, interpreting potential molecular and pathophysiological mechanisms, testing new therapeutic compounds, and guiding mechanism-specific therapy.

Publications:

Severe DCM phenotype of patient harboring RBM20 mutation S635A can be modeled by patient-specific induced pluripotent stem cell-derived cardiomyocytes.  K. Streckfuss-Bomeke , M. Tiburcy, A. Fomin., X. Luo, W. Li., C. Fischer, C. Ozcelik, A. Perrot, S. Sossalla, J. Haas, R.O. Vidal, S. Rebs, S. Khadjeh, B. Meder, S. Bonn, W.A. Linke, W.H. Zimmermann, G. Hasenfuss,  K. Guan. J Mol Cell Cardiol 2017; 113:9-21.

Establishment of an automated patch-clamp platform for electrophysiological and pharmacological evaluation of hiPSC-CMs.  W. Li*, X. Luo*, Y. Ulbricht, M. Wagner, C. Piorkowski, A. El-Armouche, K. Guan. Stem Cell Res. 2019; 41, 101662.

Electrophysiological Abnormalities in VLCAD Deficient hiPSC-Cardiomyocytes Can Be Improved by Lowering Accumulation of Fatty Acid Oxidation Intermediates.  S.J.G. Knottnerus, I. Mengarelli, R.C.I. Wust, A. Baartscheer, J.C. Bleeker, R. Coronel, S. Ferdinandusse, K. Guan, I.J. L, W. Li, X. Luo, V.M. Portero, Y. Ulbricht, G. Visser, R.J.A. Wanders, F.A. Wijburg, A:O. Verkerk, R.H. Houtkooper, C.R. Bezzina. Int J Mol Sci. 2020; 21(7).

Disease Phenotypes and Mechanisms of iPSC-Derived Cardiomyocytes From Brugada Syndrome Patients With a Loss-of-Function SCN5A Mutation.  W. Li, M. Stauske, X. Luo, S. Wagner, M. Vollrath, C.S. Mehnert, M. Schubert, L. Cyganek, S. Chen, S.M. Hasheminasab, G. Wulf, A. El-Armouche, L.S. Maier, G. Hasenfuss, K. Guan. Front Cell Dev Biol. 2020; 8, 592893.

IP3R-Mediated Compensatory Mechanism for Calcium Handling in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes With Cardiac Ryanodine Receptor Deficiency.  X. Luo *, W. Li*, K. Kunzel, S. Henze, L. Cyganek, A. Strano, M.S. Poetsch, M. Schubert, K. Guan. Front Cell Dev Biol. 2020; 8, 772.

Generation of iPSC lines from CPVT patient carrying heterozygous mutation p.A2254V in the ryanodine receptor 2 gene.  W. Li, S. Henze, X. Luo, Y. Ulbricht, A. Richter, N. Di Donato, A.A.M. Wilde, K. Guan. Stem Cell Res. 2021; 53, 102259.

Blebbistatin protects iPSC-CMs from hypercontraction and facilitates automated patch-clamp based electrophysiological study.  W. Li, X. Luo, Y. Ulbricht, K. Guan. Stem Cell Res. 2021; 56, 102565.