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Updated: Jul 12, 2025
Functional Characterization of Endogenously Expressed Human RYR1 Variants
Published on: June 9, 2021
Calcium Signaling Consequences of RyR2-S4938F Mutation Expressed in Human iPSC-Derived Cardiomyocytes
Noemi Toth1, Xiao-Hua Zhang1, Alexandra Zamaro1
1Cardiac Signaling Center, University of South Carolina, Medical University of South Carolina and Clemson University, Charleston, SC 29425, USA.
Abstract:
Type-2 ryanodine receptor (RyR2) is the major Ca2+ release channel of the cardiac sarcoplasmic reticulum (SR) that controls the rhythm and strength of the heartbeat, but its malfunction may generate severe arrhythmia leading to sudden cardiac death or heart failure. S4938F-RyR2 mutation in the carboxyl-terminal was expressed in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 gene-editing technique. Ca2+ signaling and electrophysiological properties of beating cardiomyocytes carrying the mutation were studied using total internal reflection fluorescence microscopy (TIRF) and patch clamp technique. In mutant cells, L-type Ca2+ currents (ICa), measured either by depolarizations to zero mV or repolarizations from +100 mV to -50 mV, and their activated Ca2+ transients were significantly smaller, despite their larger caffeine-triggered Ca2+ release signals compared to wild type (WT) cells, suggesting ICa-induced Ca2+ release (CICR) was compromised. The larger SR Ca2+ content of S4938F-RyR2 cells may underlie the higher frequency of spontaneously occurring Ca2+ sparks and Ca2+ transients and their arrhythmogenic phenotype.
Insights
Mutations in the type-2 ryanodine receptor (RyR2) can cause heart rhythm problems. This study found that a specific RyR2 mutation (S4938F) in human cardiomyocytes impairs calcium signaling, leading to an arrhythmogenic phenotype.
Area of Science:
- Cardiovascular Biology
- Molecular Cardiology
- Stem Cell Biology
Background:
- Type-2 ryanodine receptor (RyR2) is crucial for cardiac function, regulating calcium (Ca2+) release from the sarcoplasmic reticulum.
- RyR2 dysfunction is linked to arrhythmias, sudden cardiac death, and heart failure.
- Understanding specific RyR2 mutations is vital for developing targeted therapies.
Purpose of the Study:
- To investigate the functional consequences of the S4938F-RyR2 mutation in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs).
- To analyze the impact of this mutation on Ca2+ signaling and electrophysiological properties.
- To elucidate the underlying mechanisms of the arrhythmogenic phenotype associated with the S4938F-RyR2 mutation.
Main Methods:
- CRISPR/Cas9 gene editing was used to introduce the S4938F-RyR2 mutation into hiPSC-CMs.
- Total internal reflection fluorescence microscopy (TIRF) was employed to study Ca2+ signaling.
- Patch clamp techniques were utilized to assess electrophysiological properties, including L-type Ca2+ currents (ICa).
Main Results:
- hiPSC-CMs with the S4938F-RyR2 mutation exhibited significantly smaller L-type Ca2+ currents (ICa) and associated Ca2+ transients compared to wild-type cells.
- Mutant cells showed larger caffeine-triggered Ca2+ release, indicating increased sarcoplasmic reticulum (SR) Ca2+ content.
- These findings suggest compromised Ca2+-induced Ca2+ release (CICR) and a higher frequency of spontaneous Ca2+ events, contributing to an arrhythmogenic phenotype.
Conclusions:
- The S4938F-RyR2 mutation impairs cardiac Ca2+ handling by reducing ICa and compromising CICR.
- Increased SR Ca2+ content in mutant cells promotes spontaneous Ca2+ release, leading to arrhythmias.
- This study provides insights into the molecular mechanisms of RyR2-mediated cardiac dysfunction and potential therapeutic targets.
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