Abstract：
BACKGROUND: Variants of the SCN5A gene, which encodes the NaV1.5 cardiac sodium channel, have been linked to arrhythmic disorders associated with dilated cardiomyopathy (DCM). However, the precise pathological mechanisms remain elusive. The present study aimed to elucidate the pathophysiological consequences of the DCM-linked Nav1.5/R219H variant, which is known to generate a gating pore current, using patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in monolayers.
METHODS: Ventricular- and atrial-like hiPSC-CM monolayers were generated from DCM patients carrying the R219H SCN5A variant as well as from healthy control individuals. CRISPR-corrected hiPSC-CMs served as isogenic controls. Simultaneous optical mapping of action potentials (APs) and calcium transients (CaTs) was employed to measure conduction velocities (CVs) and AP durations (APDs) and served as markers of electrical excitability. Calcium handling was evaluated by assessing CaT uptake (half-time to peak), recapture (tau of decay), and durations (TD50 and TD80). A multi-electrode array (MEA) analysis was conducted on hiPSC-CM monolayers to measure field potential (FP) parameters, including corrected Fridericia FP durations (FPDc).
RESULTS: Our results revealed that CVs were significantly reduced by more than 50 % in both ventricular- and atrial-like hiPSC-CM monolayers carrying the R219H variant compared to the control group. APDs were also prolonged in the R219H group compared to the control and CRISPR-corrected groups. CaT uptake, reuptake, and duration were also markedly delayed in the R219H group compared to the control and CRISPR-corrected groups in both the ventricular- and the atrial-like hiPSC-CM monolayers. Lastly, the MEA data revealed a notably prolonged FPDc in the ventricular- and atrial-like hiPSC-CMs carrying the R219H variant compared to the control and isogenic control groups.
CONCLUSIONS: These findings highlight the impact of the gating pore current on AP propagation and calcium homeostasis within a functional syncytium environment and offer valuable insights into the potential mechanisms underlying DCM pathophysiology.
METHODS: Ventricular- and atrial-like hiPSC-CM monolayers were generated from DCM patients carrying the R219H SCN5A variant as well as from healthy control individuals. CRISPR-corrected hiPSC-CMs served as isogenic controls. Simultaneous optical mapping of action potentials (APs) and calcium transients (CaTs) was employed to measure conduction velocities (CVs) and AP durations (APDs) and served as markers of electrical excitability. Calcium handling was evaluated by assessing CaT uptake (half-time to peak), recapture (tau of decay), and durations (TD50 and TD80). A multi-electrode array (MEA) analysis was conducted on hiPSC-CM monolayers to measure field potential (FP) parameters, including corrected Fridericia FP durations (FPDc).
RESULTS: Our results revealed that CVs were significantly reduced by more than 50 % in both ventricular- and atrial-like hiPSC-CM monolayers carrying the R219H variant compared to the control group. APDs were also prolonged in the R219H group compared to the control and CRISPR-corrected groups. CaT uptake, reuptake, and duration were also markedly delayed in the R219H group compared to the control and CRISPR-corrected groups in both the ventricular- and the atrial-like hiPSC-CM monolayers. Lastly, the MEA data revealed a notably prolonged FPDc in the ventricular- and atrial-like hiPSC-CMs carrying the R219H variant compared to the control and isogenic control groups.
CONCLUSIONS: These findings highlight the impact of the gating pore current on AP propagation and calcium homeostasis within a functional syncytium environment and offer valuable insights into the potential mechanisms underlying DCM pathophysiology.
摘要：
背景：SCN5A基因的变异体，它编码NaV1.5心脏钠通道，已与扩张型心肌病（DCM）相关的心律失常有关。然而,精确的病理机制仍然难以捉摸。本研究旨在阐明DCM连接的Nav1.5/R219H变体的病理生理后果，已知会产生门控孔电流，使用患者特异性人诱导多能干细胞衍生的心肌细胞（hiPSC-CM）在单层中培养。
方法：脑室和心房样hiPSC-CM单层是从携带R219HSCN5A变体的DCM患者以及健康对照个体中产生的。CRISPR校正的hiPSC-CM用作等基因对照。动作电位（AP）和钙瞬变（CaT）的同时光学映射用于测量传导速度（CV）和AP持续时间（APD），并用作电兴奋性的标记。通过评估CaT摄取(达到峰值的一半时间)来评估钙处理,重新捕获（tau的衰变），和持续时间（TD50和TD80）。在hiPSC-CM单层上进行多电极阵列（MEA）分析，以测量场电位（FP）参数，包括校正的FridericiaFP持续时间（FPDc）。
结果：我们的结果表明，与对照组相比，携带R219H变体的心室和心房样hiPSC-CM单层的CV显着降低了50％以上。与对照组和CRISPR校正组相比，R219H组的APD也延长了。CaT吸收,再摄取,在心室和心房样hiPSC-CM单层中，与对照组和CRISPR校正组相比，R219H组的持续时间也明显延迟。最后,MEA数据显示,与对照组和等基因对照组相比,携带R219H变异体的脑室和心房样hiPSC-CM的FPDc显著延长.
结论：这些发现强调了门控孔电流对功能性合胞体环境中AP增殖和钙稳态的影响，并为DCM病理生理学潜在机制提供了有价值的见解。
方法：脑室和心房样hiPSC-CM单层是从携带R219HSCN5A变体的DCM患者以及健康对照个体中产生的。CRISPR校正的hiPSC-CM用作等基因对照。动作电位（AP）和钙瞬变（CaT）的同时光学映射用于测量传导速度（CV）和AP持续时间（APD），并用作电兴奋性的标记。通过评估CaT摄取(达到峰值的一半时间)来评估钙处理,重新捕获（tau的衰变），和持续时间（TD50和TD80）。在hiPSC-CM单层上进行多电极阵列（MEA）分析，以测量场电位（FP）参数，包括校正的FridericiaFP持续时间（FPDc）。
结果：我们的结果表明，与对照组相比，携带R219H变体的心室和心房样hiPSC-CM单层的CV显着降低了50％以上。与对照组和CRISPR校正组相比，R219H组的APD也延长了。CaT吸收,再摄取,在心室和心房样hiPSC-CM单层中，与对照组和CRISPR校正组相比，R219H组的持续时间也明显延迟。最后,MEA数据显示,与对照组和等基因对照组相比,携带R219H变异体的脑室和心房样hiPSC-CM的FPDc显著延长.
结论：这些发现强调了门控孔电流对功能性合胞体环境中AP增殖和钙稳态的影响，并为DCM病理生理学潜在机制提供了有价值的见解。
