Department of Molecular Pharmacology and Experimental Therapeutics (S.I.E., D.Y., W.Z., S.M.D., D.J.T., J.M.B., C.S.J.K., M.J.A.).
Windland Smith Rice Sudden Death Genomics Laboratory (S.I.E., D.Y., W.Z., S.M.D., D.J.T., J.M.B., C.S.J.K., M.J.A.).
Circ Genom Precis Med. 2019 Aug;12(8):e002534. doi: 10.1161/CIRCGEN.119.002534. Epub 2019 Aug 20.
The CACNA1C-encoded cardiac L-type calcium channel (Cav1.2) is essential for cardiocyte action potential duration (APD). We previously reported the CACNA1C-p.R518C variant associated with prolonged QT intervals, cardiomyopathy, and sudden cardiac death in several pedigrees.
To characterize a patient-derived human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) CACNA1C-p.R518C model, CACNA1C-p.R518C hiPSC-CMs were generated from a 13-year-old man (QTc, >480 ms) with a family history of sudden cardiac death. An isogenic hiPSC-CM gene-corrected control was created using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9). APD and calcium handling were assessed by live cell imaging with Arclight voltage and Fluo-4 calcium indicators, respectively. The APD and L-type calcium channel biophysical properties were further assessed by whole-cell patch clamp technique.
The Bazett formula-corrected, Arclight-measured APD of CACNA1C-p.R518C hiPSC-CMs was significantly longer (622±11 ms; n=92) than the isogenic control hiPSC-CMs (453±5 ms; n=62; P<0.0001). Patch clamp assessment of CACNA1C-p.R518C hiPSC-CMs paced at 1 Hz confirmed a prolonged APD (689±29 ms; n=10) compared with the patient's isogenic control hiPSC-CMs (434±30 ms; n=8; P<0.05). Fluo-4-measured calcium transient decay time suggested calcium mishandling in CACNA1C-p.R518C. Patch clamp analysis revealed increased L-type calcium channel window current, slow decay time at various voltages, and increased late calcium current for CACNA1C-p.R518C hiPSC-CMs when compared with isogenic control hiPSC-CMs.
Using patient-specific hiPSC-CM mutant and isogenic control lines, we demonstrate that the CACNA1C-p.R518C variant is the self-sufficient, monogenetic substrate for the patient's long-QT syndrome phenotype. These data further bolster the conclusion that CACNA1C is a bona fide, definite evidence long-QT syndrome susceptibility gene.
CACNA1C 编码的心脏 L 型钙通道（Cav1.2）对心肌细胞动作电位持续时间（APD）至关重要。我们之前报道了 CACNA1C-p.R518C 变体与多个家系的 QT 间期延长、心肌病和心脏性猝死相关。
为了对源自患者的诱导多能干细胞衍生的心肌细胞（hiPSC-CM）CACNA1C-p.R518C 模型进行表征，我们从一名 13 岁男性（QTc>480ms）中生成了携带 CACNA1C-p.R518C 变体的 hiPSC-CM，该男性有心脏性猝死的家族史。使用 CRISPR/Cas9（成簇规律间隔短回文重复/成簇规律间隔短回文重复相关 9）创建了同源性 hiPSC-CM 基因校正对照。通过使用 Arclight 电压和 Fluo-4 钙指示剂分别进行活细胞成像，评估 APD 和钙处理。通过全细胞膜片钳技术进一步评估 APD 和 L 型钙通道生物物理特性。
Bazett 公式校正的、Arclight 测量的 CACNA1C-p.R518C hiPSC-CM 的 APD 明显更长（622±11ms；n=92），而同源性 hiPSC-CM 的 APD 更短（453±5ms；n=62；P<0.0001）。以 1Hz 起搏时对 CACNA1C-p.R518C hiPSC-CM 的全细胞膜片钳评估证实 APD 延长（689±29ms；n=10），而与患者的同源性 hiPSC-CM 相比（434±30ms；n=8；P<0.05）。Fluo-4 测量的钙瞬变衰减时间提示 CACNA1C-p.R518C 钙处理异常。与同源性 hiPSC-CM 相比，全细胞膜片钳分析显示 CACNA1C-p.R518C hiPSC-CM 的 L 型钙通道窗口电流增加、在不同电压下衰减时间变慢、晚期钙电流增加。
使用患者特异性 hiPSC-CM 突变体和同源性 hiPSC-CM 对照系，我们证明 CACNA1C-p.R518C 变体是患者长 QT 综合征表型的自足的、单基因基础。这些数据进一步证实 CACNA1C 是真正的、明确的长 QT 综合征易感性基因。
