Mutating RyR2 Ca2+-binding residues in human cardiomyocytes suppressed ICa- and caffeine-triggered Ca2+ releases, with only 20% to 70% of intact mutant myocytes responding to caffeine.
Mutations in RyR2 Ca2+-binding residues suppress SR Ca2+ release but allow spontaneous beating to persist, suggesting remodelling of excitation-contraction coupling pathways.
AIMS: CRISPR/Cas9 gene edits of cardiac ryanodine receptor (RyR2) in human-induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) provide a novel platform for introducing mutations in RyR2 Ca2+-binding residues and examining the resulting excitation contraction (EC)-coupling remodelling consequences. METHODS AND RESULTS: Ca2+-signalling phenotypes of mutations in RyR2 Ca2+-binding site residues associated with cardiac arrhythmia (RyR2-Q3925E) or not proven to cause cardiac pathology (RyR2-E3848A) were determined using ICa- and caffeine-triggered Ca2+ releases in voltage-clamped and total internal reflection fluorescence-imaged wild type and mutant cardiomyocytes infected with sarcoplasmic reticulum (SR)-targeted ER-GCaMP6 probe. (i) ICa- and caffeine-triggered Fura-2 or ER-GCaMP6 signals were suppressed, even when ICa was significantly enhanced in Q3925E and E3848A mutant cardiomyocytes; (ii) spontaneous beating (Fura-2 Ca2+ transients) persisted in mutant cells without the SR-release signals; (iii) while 5-20 mM caffeine failed to trigger Ca2+-release in voltage-clamped mutant cells, only ∼20% to ∼70% of intact myocytes responded respectively to caffeine; (iv) and 20 mM caffeine transients, however, activated slowly, were delayed, and variably suppressed by 2-APB, FCCP, or ruthenium red. CONCLUSION: Mutating RyR2 Ca2+-binding residues, irrespective of their reported pathogenesis, suppressed both ICa- and caffeine-triggered Ca2+ releases, suggesting interaction between Ca2+- and caffeine-binding sites. Enhanced transmembrane calcium influx and remodelling of EC-coupling pathways may underlie the persistence of spontaneous beating in Ca2+-induced Ca2+ release-suppressed mutant myocytes.
Xia et al. (Fri,) conducted a other in Cardiac arrhythmia. CRISPR/Cas9 gene edits of RyR2 Ca2+-binding residues (Q3925E and E3848A) vs. Wild type cardiomyocytes was evaluated on Ca2+-signalling phenotypes (ICa- and caffeine-triggered Ca2+ releases). Mutating RyR2 Ca2+-binding residues in human cardiomyocytes suppressed ICa- and caffeine-triggered Ca2+ releases, with only 20% to 70% of intact mutant myocytes responding to caffeine.