Cardiac Signaling Center of University of South Carolina, Medical University of South Carolina and Clemson University, 68 President Street, Bioengineering building Rm 306, Charleston, SC 29425, USA.
Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, 68 President Street, Bioengineering building Rm 306, Charleston, SC 29425, USA.
Cardiovasc Res. 2024 Feb 27;120(1):44-55. doi: 10.1093/cvr/cvad163.
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.
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.
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.
在人诱导多能干细胞衍生的心肌细胞（hiPSC-CM）中，使用 CRISPR/Cas9 基因编辑心脏兰尼碱受体（RyR2），为引入 RyR2 Ca2+结合残基突变并研究由此产生的兴奋-收缩（EC）偶联重塑后果提供了一个新平台。
使用电压钳位和全内反射荧光成像的野生型和突变型心肌细胞中的 ICa 和咖啡因触发的 Ca2+释放，来确定与心律失常相关的 RyR2 Ca2+结合位点残基（RyR2-Q3925E）或未证明引起心脏病理学的突变（RyR2-E3848A）的 Ca2+信号表型。（i）在 Q3925E 和 E3848A 突变型心肌细胞中，即使 ICa 显著增强，ICa 和咖啡因触发的 Fura-2 或 ER-GCaMP6 信号也受到抑制；（ii）在突变细胞中，尽管没有 SR 释放信号，但自发性搏动（Fura-2 Ca2+瞬变）仍然存在；（iii）虽然 5-20mM 咖啡因未能在电压钳制的突变细胞中触发 Ca2+释放，但只有约 20%到约 70%的完整心肌细胞分别对咖啡因产生反应；（iv）然而，20mM 咖啡因瞬变缓慢激活，被 2-APB、FCCP 或钌红延迟和可变抑制。
突变 RyR2 Ca2+结合残基，无论其报道的发病机制如何，都抑制了 ICa 和咖啡因触发的 Ca2+释放，提示 Ca2+和咖啡因结合位点之间的相互作用。增强的跨膜钙内流和 EC 偶联途径的重塑可能是 Ca2+诱导的 Ca2+释放抑制后突变型肌细胞中自发性搏动持续存在的基础。
