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Updated: Jun 3, 2025
Functional Characterization of Endogenously Expressed Human RYR1 Variants
Published on: June 9, 2021
Attempts to Create Transgenic Mice Carrying the Q3924E Mutation in RyR2 Ca2+ Binding Site
Xiao-Hua Zhang1, Fu-Lei Tang2, Allison M Trouten3
1Cardiac Signaling Center, University of South Carolina, Medical University of South Carolina and Clemson University, Charleston, SC 29425, USA.
Abstract:
Over 200 point mutations in the ryanodine receptor (RyR2) of the cardiac sarcoplasmic reticulum (SR) are known to be associated with cardiac arrhythmia. We have already reported on the calcium signaling phenotype of a point mutation in RyR2 Ca2+ binding site Q3925E expressed in human stem-cell-derived cardiomyocytes (hiPSC-CMs) that was found to be lethal in a 9-year-old girl. CRISPR/Cas9-gene-edited mutant cardiomyocytes carrying the RyR2-Q3925E mutation exhibited a loss of calcium-induced calcium release (CICR) and caffeine-triggered calcium release but continued to beat arrhythmically without generating significant SR Ca2+ release, consistent with a remodeling of the calcium signaling pathway. An RNAseq heat map confirmed significant changes in calcium-associated genes, supporting the possibility of remodeling. To determine the in situ cardiac phenotype in an animal model of this mutation, we generated a knock-in mouse model of RyR2-Q3924E+/- using the CRISPR/Cas9 technique. We obtained three homozygous and one chimera mice, but they all died before reaching 3 weeks of age, preventing the establishment of germline mutation transmission in their offspring. A histo-pathological analysis of the heart showed significant cardiac hypertrophy, suggesting the Q3924E-RyR2 mutation was lethal to the mice.
Insights
Mutations in the ryanodine receptor (RyR2) cause cardiac arrhythmia. A lethal RyR2 mutation (Q3925E) in a child led to a mouse model exhibiting early death and cardiac hypertrophy, confirming its lethality.
Area of Science:
- Cardiovascular Research
- Molecular Cardiology
- Genetics
Background:
- Over 200 point mutations in the cardiac ryanodine receptor (RyR2) are linked to cardiac arrhythmias.
- A previously identified RyR2 Q3925E mutation was lethal in a pediatric patient.
Purpose of the Study:
- To investigate the in situ cardiac phenotype of the RyR2-Q3925E mutation in a mouse model.
- To determine the lethality and pathological effects of this specific RyR2 mutation in vivo.
Main Methods:
- CRISPR/Cas9 gene editing was used to create RyR2-Q3924E+/- knock-in mice.
- Homozygous and chimeric mice were analyzed for survival and cardiac pathology.
- Histopathological analysis was performed on affected hearts.
Main Results:
- All generated RyR2-Q3924E mutant mice (homozygous and chimeric) died before 3 weeks of age.
- Histopathological examination revealed significant cardiac hypertrophy in the affected mice.
- The mutation prevented germline transmission due to early lethality.
Conclusions:
- The RyR2-Q3924E mutation is embryonically lethal in mice, manifesting as severe cardiac hypertrophy.
- This animal model confirms the critical role of RyR2 function in early cardiac development and survival.
- The findings underscore the severe consequences of RyR2 dysfunction in inherited cardiac arrhythmias.
