IP3R-Mediated Compensatory Mechanism for Calcium Handling in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes With Cardiac Ryanodine Receptor Deficiency

Xiaojing Luo; Wener Li; Karolina Künzel; Sarah Henze; Lukas Cyganek; Anna Strano; Mareike S Poetsch; Mario Schubert; Kaomei Guan

doi:10.3389/fcell.2020.00772

IP3R-Mediated Compensatory Mechanism for Calcium Handling in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes With Cardiac Ryanodine Receptor Deficiency

Front Cell Dev Biol. 2020 Aug 12:8:772. doi: 10.3389/fcell.2020.00772. eCollection 2020.

Authors

Xiaojing Luo¹, Wener Li¹, Karolina Künzel¹, Sarah Henze², Lukas Cyganek^{2

3}, Anna Strano¹, Mareike S Poetsch¹, Mario Schubert¹, Kaomei Guan^{1

2}

Affiliations

¹ Institute of Pharmacology and Toxicology, Technische Universität Dresden, Dresden, Germany.
² Clinic for Cardiology and Pneumology, Universitätsmedizin Göttingen, Göttingen, Germany.
³ DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany.

Abstract

In adult cardiomyocytes (CMs), the type 2 ryanodine receptor (RYR2) is an indispensable Ca²⁺ release channel that ensures the integrity of excitation-contraction coupling, which is fundamental for every heartbeat. However, the role and importance of RYR2 during human embryonic cardiac development are still poorly understood. Here, we generated two human induced pluripotent stem cell (iPSC)-based RYR2 knockout (RYR2^-/-) lines using the CRISPR/Cas9 gene editing technology. We found that RYR2^-/--iPSCs could differentiate into CMs with the 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, we observed significantly reduced frequency and intense abnormalities of Ca²⁺ transients in RYR2^-/--iPSC-CMs. Ctrl-iPSC-CMs displayed sensitivity to extracellular Ca²⁺ ([Ca²⁺ ]_o) and caffeine in a concentration-dependent manner, while RYR2^-/--iPSC-CMs showed inconsistent reactions to [Ca²⁺ ]_o and were insensitive to caffeine, indicating there is no RYR2-mediated Ca²⁺ release from the sarcoplasmic reticulum (SR). Instead, compensatory mechanism for calcium handling in RYR2^-/--iPSC-CMs is partially mediated by the inositol 1,4,5-trisphosphate receptor (IP3R). Similar to Ctrl-iPSC-CMs, SR Ca²⁺ refilling in RYR2^-/--iPSC-CMs is mediated by SERCA. Additionally, RYR2^-/--iPSC-CMs showed a decreased beating rate and a reduced peak amplitude of L-type Ca²⁺ current. These findings demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function. IP3R-mediated Ca²⁺ release is one of the major compensatory mechanisms for Ca²⁺ cycling in human CMs with the RYR2 deficiency.

Keywords: CRISPR/Cas9; calcium handling; induced pluripotent stem cell-derived cardiomyocytes; inositol 1,4,5-trisphosphate receptor; ryanodine receptor 2.