Purkinje cells from RyR2 mutant mice are highly arrhythmogenic but responsive to targeted therapy.
Purkinje cells from RyR2 mutant mice are highly arrhythmogenic but responsive to targeted therapy.
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DOI：
10.1161/circresaha.110.221481
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发表时间：
2010-08-20
影响因子：
20.1
通讯作者：
Fishman GI
中科院分区：
文献类型：
--
作者：
Kang G;Giovannone SF;Liu N;Liu FY;Zhang J;Priori SG;Fishman GI
浦肯野纤维网络被认为是儿茶酚胺能性多形性室性心动过速（CPVT）中促心律失常性Ca 2+释放事件的来源，但缺乏在细胞水平支持这一机制的证据。我们试图确定自发性Ca 2+释放事件的频率和严重程度，以及RyR 2 R4496 C/+ CPVT突变小鼠和同窝对照小鼠的浦肯野细胞和心室肌细胞对抗心律失常药物氟卡尼的反应。我们将RyR 2 R4496 C/+基因敲入小鼠与新描述的Cntn 2-EGFP BAC转基因小鼠杂交，Cntn 2-EGFP BAC转基因小鼠在心脏传导系统的细胞中表达荧光报告基因，包括远端浦肯野纤维网络。分离的VM（EGFP-）和浦肯野细胞（EGFP+）从野生型心脏和突变的心脏区分落射荧光和细胞内Ca 2+动态记录的显微荧光。与相同基因型的心室肌细胞相比，野生型和RyR 2 R4496 C/+突变体浦肯野细胞均显示出显著较慢的激活和舒张动力学，并且突变体浦肯野细胞中的τ衰减显著慢于野生型浦肯野细胞中观察到的τ衰减。在研究的四组中，RyR 2 R4496 C/+突变体浦肯野细胞也最有可能发生自发性Ca 2+释放事件，每个细胞的事件数量也显著更多。此外，用异丙肾上腺素处理，虽然所有四组均显示出致炎性Ca 2 +i事件的频率增加，但RyR 2 R4496 C/+浦肯野细胞对细胞内Ca 2+处理的最显著异常作出反应，包括未刺激的Ca 2 +i事件的频率显著增加、交替的发展以及孤立和持续的触发性搏动。来自野生型小鼠的浦肯野细胞和心室肌细胞均表现出氟卡尼对自发性Ca 2+释放事件的抑制，而在RyR 2 R4496 C/+小鼠中，浦肯野细胞优先对药物反应。相反，RyR 2阻断剂丁卡因在突变型浦肯野细胞和心室肌细胞中同样有效。浦肯野细胞比心室肌细胞显示出更大的倾向，发展异常的细胞内Ca 2+处理。这种促心律失常行为通过RyR 2 Ca 2+释放通道中的致病突变而增强，并通过儿茶酚胺能刺激而大大加剧，并发展促心律失常触发搏动。这些数据支持浦肯野细胞是CPVT动物模型和人类中触发心律失常的关键因素的概念，并表明浦肯野纤维网络在室性心律失常的发生中具有更广泛的作用。
The Purkinje fiber network has been proposed as the source of arrhythmogenic Ca2+ release events in catecholaminergic polymorphic ventricular tachycardia (CPVT) yet evidence supporting this mechanism at the cellular level is lacking. We sought to determine the frequency and severity of spontaneous Ca2+ release events and the response to the anti-arrhythmic agent flecainide in Purkinje cells and ventricular myocytes from RyR2R4496C/+ CPVT mutant mice and littermate controls. We crossed RyR2R4496C/+ knock-in mice with the newly described Cntn2-EGFP BAC transgenic mice, which express a fluorescent reporter gene in cells of the cardiac conduction system, including the distal Purkinje fiber network. Isolated VMs (EGFP-) and Purkinje cells (EGFP+) from wild type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca2+ dynamics recorded by microfluorimetry. Both wild type and RyR2R4496C/+ mutant Purkinje cells displayed significantly slower kinetics of activation and relaxation compared to ventricular myocytes of the same genotype, and τdecay in the mutant Purkinje cells was significantly slower than that observed in wild type Purkinje cells. Of the four groups studied, RyR2R4496C/+ mutant Purkinje cells were also most likely to develop spontaneous Ca2+ release events and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, while all four groups showed increases in the frequency of arrhythmogenic Ca2+i events, the RyR2R4496C/+ Purkinje cells responded with the most profound abnormalities in intracellular Ca2+ handling, including a significant increase in the frequency of unstimulated Ca2+i events, the development of alternans, as well as isolated and sustained runs of triggered beats. Both Purkinje cells and ventricular myocytes from wild type mice showed suppression of spontaneous Ca2+ release events with flecainide, whereas in RyR2R4496C/+ mice, the Purkinje cells were preferentially responsive to drug. In contrast, the RyR2 blocker tetracaine was equally efficacious in mutant Purkinje cells and ventricular myocytes. Purkinje cells display a greater propensity to develop abnormalities in intracellular Ca2+ handling than ventricular myocytes. This pro-arrhythmic behavior is enhanced by disease-causing mutations in the RyR2 Ca2+ release channel and greatly exacerbated by catecholaminergic stimulation, with the development of arrhythmogenic triggered beats. These data support the concept that Purkinje cells are critical contributors to arrhythmic triggers in animal models and humans with CPVT and suggest a broader role for the Purkinje fiber network in the genesis of ventricular arrhythmias.