Enhanced binding of calmodulin to RyR2 corrects arrhythmogenic channel disorder in CPVT-associated myocytes
Enhanced binding of calmodulin to RyR2 corrects arrhythmogenic channel disorder in CPVT-associated myocytes
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DOI：
10.1016/j.bbrc.2014.03.152
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发表时间：
2014-05-23
影响因子：
3.1
通讯作者：
Yano, Masafumi
中科院分区：
文献类型：
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作者：
Fukuda, Masakazu;Yamamoto, Takeshi;Yano, Masafumi
目的：钙调蛋白 (CaM) 在调节兰尼碱受体 (RyR2) 通道门控中发挥关键作用。在这里，我们使用具有 CPVT 相关 RyR2 突变 (R24745) 的敲入 (RI) 小鼠模型，研究了 (a) CaM 在 CPVT 通道障碍中的致病作用，以及 (b) 纠正 CPVT 相关通道障碍的可能性。方法和结果：在离体心室肌细胞起搏 (1-5 Hz) 之前和之后以全细胞电流模式记录跨膜电位。通过在皂苷通透的肌细胞中掺入用 HiLyte Fluor (R) 荧光标记的外源 CaM 来评估 CaM 结合。在 cAMP (1 μM) 存在下，IC 细胞中 CaM 与 RyR 结合的表观亲和力降低 (Kd: 140-400 nM)，但在 WT 细胞中则没有降低 (Kd: 110-120 nM)。 Gly-Ser-His-CaM（GSH-CaM 的 RyR 结合力比 CaM 更高）恢复了与 cAMP 处理的 KI 细胞 (140 nM) 的 RyR 的正常结合。即使在 5 Hz 起搏下，WT 细胞中也未观察到延迟后除极 (DAD) 和触发活动 (TA)，而 IU 细胞中分别有 20% 和 12% 观察到 DAD 和 TA。响应 10 nM 异丙肾上腺素，在 11% 的 WT 细胞中仅观察到 DAD（但未观察到 TA），而在 KI 细胞中，DAD 和 TA 的发生率进一步分别增加至细胞的 60% 和 38%。向 RI 细胞中添加 GSH-CaM (100 nM) 会降低 DAD 和 TA（DAD：38% 的细胞；TA：10% 的细胞），而 CaM (100 nM) 则没有明显效果。向皂苷通透的 IC 细胞中添加 GSH-CaM 会降低 Ca2+ 火花频率（+33% WT 细胞），而没有 GSH-CaM 则显着增加（+100% WT 细胞），而 CaM 对 Ca2+ 火花频率的影响要小得多（+76% WT 细胞）。然后，通过将 CaM 或 GSH-CaM 掺入完整细胞（使用蛋白质递送试剂盒），我们评估了 GSH-CaM（胞质 [CaM] = 240 nM，胞质 [GSH-CaM] = 230 nM）对自发 Ca2+ 瞬变频率（sCaT，占细胞总数的百分比）的原位效应。在 ICI 细胞中添加 10 nM 异丙肾上腺素会增加短暂 5 Hz 起搏后的 sCaT (37%)，而 GSH-CaM (9%) 比 CaM (26%) 更能减弱 sCaT（P < 0.01 vs CaM）。结论：CPVT 突变细胞的 RyR 通道功能特征存在多种紊乱（自发 Ca2+ 渗漏增加，延迟通过增加 CaM 与 RyR 结合的亲和力，可以将后去极化、触发活动、Ca2+ 火花频率、自发 Ca2+ 瞬变）校正为正常功能。 (C) 2014 Elsevier Inc. 保留所有权利。
Aims: Calmodulin (CaM) plays a key role in modulating channel gating in ryanodine receptor (RyR2). Here, we investigated (a) the pathogenic role of CaM in the channel disorder in CPVT and (b) the possibility of correcting the CPVT-linked channel disorder, using knock-in (RI) mouse model with CPVT-associated RyR2 mutation (R24745).Methods and results: Transmembrane potentials were recorded in whole cell current mode before and after pacing (1-5 Hz) in isolated ventricular myocytes. CaM binding was assessed by incorporation of exogenous CaM fluorescently labeled with HiLyte Fluor (R) in saponin-permeabilized myocytes. In the presence of cAMP (1 mu M) the apparent affinity of CaM binding to the RyR decreased in IC cells (Kd: 140-400 nM), but not in WT cells (Kd: 110-120 nM). Gly-Ser-His-CaM (GSH-CaM that has much higher RyR-binding than CaM) restored normal binding to the RyR of cAMP-treated KI cells (140 nM). Neither delayed afterdepolarization (DAD) nor triggered activity (TA) were observed in WT cells even at 5 Hz pacing, whereas both DAD and TA were observed in 20% and 12% of IU cells, respectively. In response to 10 nM isoproterenol, only DAD (but not TA) was observed in 11% of WT cells, whereas in KI cells the incidence of DAD and TA further increased to 60% and 38% of cells, respectively. Addition of GSH-CaM (100 nM) to RI cells decreased both DADs and TA (DAD: 38% of cells; TA: 10% of cells), whereas CaM (100 nM) had no appreciable effect. Addition of GSH-CaM to saponin-permeabilized IC cells decreased Ca2+ spark frequency (+33% of WT cells), which otherwise markedly increased without GSH-CaM (+100% of WT cells), whereas CaM revealed much less effect on the Ca2+ spark frequency (+76% of WT cells). Then, by incorporating CaM or GSH-CaM to intact cells (with protein delivery kit), we assessed the in situ effect of GSH-CaM (cytosolic [CaM] = 240 nM, cytosolic [GSH-CaM] = 230 nM) on the frequency of spontaneous Ca2+ transient (sCaT, % of total cells). Addition of 10 nM isoproterenol to ICI cells increased sCaT after transient 5 Hz pacing (37%), whereas it was much more attenuated by GSH-CaM (9%) than by CaM (26%) (P < 0.01 vs CaM).Conclusions: Several disorders in the RyR channel function characteristic of the CPVT-mutant cells (increased spontaneous Ca2+ leak, delayed afterdepolarization, triggered activity, Ca2+ spark frequency, spontaneous Ca2+ transients) can be corrected to a normal function by increasing the affinity of CaM binding to the RyR. (C) 2014 Elsevier Inc. All rights reserved.