Attempts to create transgenic mice carrying the Q3925E mutation in RyR2 Ca2+ binding site

Over 200 point mutations in ryanodine receptor (RyR2) of cardiac sarcoplasmic reticulum (SR) are known to associate 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 RyR2-Q3925E mutation exhibited 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 remodeling of calcium signaling pathway. RNAseq heat-map confirmed significant changes of calcium associated genes, supporting the remodeling possibility. To determine the in-situ cardiac phenotype in animal model of this mutation, we generated a knock-in mouse model of RyR2-Q3924E+/– using CRISPR/Cas9 technique. We obtained 3 homozygous and one chimera mice, but they all died before 3 weeks of age preventing the establishment of germline mutation transmission in the offsprings. Histo-pathological analysis of the heart showed significant cardiac hypertrophy suggesting Q3924E-RyR2 mutation was lethal in the mouse.