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

Abstract

In adult cardiomyocytes (CMs), the type 2 ryanodine receptor (RYR2) is an indispensable Ca 2+ 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 Ca 2+ handling, we observed significantly reduced frequency and intense abnormalities of Ca 2+ transients in RYR2 −/−-iPSC-CMs. Ctrl-iPSC-CMs displayed sensitivity to extracellular Ca 2+ ([Ca 2+ ] o) and caffeine in a concentration-dependent manner, while RYR2 −/−-iPSC-CMs showed inconsistent reactions to [Ca 2+ ] o and were insensitive to caffeine, indicating there is no RYR2mediated Ca 2+ 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 Ca 2+ 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 Ca 2+ current. These findings demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function. IP3R-mediated Ca 2+ release is one of the major compensatory mechanisms for Ca 2+ cycling in human CMs with the RYR2 deficiency.

Disease Phenotypes and Mechanisms of iPSC-Derived Cardiomyocytes From Brugada Syndrome Patients With a Loss-of-Function SCN5A Mutation

“…The impulse protocol for I CaL was shown in inset of Figures 4H,I,K, as previously described (Luo et al, 2020). The current-voltage (I-V) impulse depolarized from −90 to −80 mV then every 10 mV as step to +70 mV with a duration of 600 ms. Steady-state inactivation was measured by using a two-pulse protocol at a holding potential of −90 mV.…”

Calpain cleavage of Junctophilin-2 generates a spectrum of calcium-dependent cleavage products and DNA-rich NT1-fragment domains in cardiomyocytes

“…Since JP2 is very important for the maintenance of the cardiac dyad structure, we wondered how loss-of-function of RyR2 affects JP2 expression and cleavage. Therefore, we applied RyR2 knockout iPSC lines that were generated using the CRISPR/Cas9 gene editing technology 37 . In RyR2 knockout hiPSC-CMs, CICR is completely abolished, with accompanying impaired contractility and cell survival 37 .…”

Blebbistatin protects iPSC-CMs from hypercontraction and facilitates automated patch-clamp based electrophysiological study

“…Paced whole-cell calcium transients were measured according to our previous publication (Luo et al, 2020). CMs around day 80 were dissociated and replated on coverslips at a density of 200,000 cells/well (6 well plate).…”