Pluripotent stem cells, derived from skin fibroblasts of two relatives with familial long-QT syndrome and induced to differentiate into cardiac myocytes, exhibited the electrophysiologic traits of the disorder, showing for the first time that stem cell models “can recapitulate aspects of genetic cardiac diseases,” according to a small study reported in the Oct. 7 issue of the New England Journal of Medicine.
The research findings have far-reaching implications for cardiac research in general, not just for the relatively uncommon long-QT syndrome, including opening the door to in vitro development and testing of new cardiac medications, said Alessandra Moretti, Ph.D., and her associates at the Technical University of Munich.
They obtained skin fibroblasts from an 8-year-old boy when clinical evaluation for attention-deficit/hyperactivity disorder revealed a prolonged QT interval on his electrocardiogram, and subsequent genotyping revealed that he carried a mutation of the KSNQ1 gene that characterizes long-QT syndrome type 1. Skin fibroblasts also were obtained from the patient’s father, who also carried the same mutation.
The researchers grew pluripotent stem cell colonies from these cells, as well as from the cells of two healthy control subjects. The cells were induced to differentiate into the cardiac lineages. Then spontaneously beating cells were subjected to pacing, then separated into three distinct types of action potentials corresponding to “ventricular,” “atrial,” and “nodal” myocytes.
When compared with myocytes derived from the control subjects, the patients’ myocytes showed prolongation of the action potential, altered IKs activation and deactivation, and an abnormal response to catecholamine stimulation – all of which are characteristic of the cardiac abnormalities in long-QT syndrome. Further characterization of the KSNQ1 mutation showed that “a dominant negative trafficking defect” was associated with the 70%-80% reduction in IKs current and altered properties governing channel activation and deactivation.
The investigators then assessed the effects of adrenergic stimulation by exposing the beating myocytes to isoproterenol. This shortened the action potential of the myocytes from the control subjects but lengthened that of the myocytes from affected patients, “exacerbating the long-QT syndrome type 1 phenotype and increasing the risk of arrhythmic events” in the cells.
Pretreating the myocytes by exposing them to the beta-blocker propranolol induced a protective effect, much as beta-blocker therapy protects affected patients from the effects of abnormally prolonged ventricular repolarization phase: a propensity for polymorphic ventricular tachycardia and sudden cardiac death.
“Our findings suggest that there may be alternative approaches to the development of candidate drugs” to treat this and other genetic cardiac disorders, Dr. Moretti and her colleagues said (N. Engl. J. Med. 2010;363:1397-409).
In particular, “the observed protective effects of beta-blockade show that it is possible to investigate the therapeutic action of medications for treating human cardiac disease in vitro with the use of patient-specific cells. This approach is particularly attractive because of the pluripotent nature of these cells and the potentially unlimited number of induced cardiomyocytes available for high-throughput drug development,” they noted.
Until now, research on the pathogenesis of the long-QT syndrome has relied primarily on genetic animal models. But because of the differences among species in the chemical channels that generate cardiac repolarizing currents, “none of the available [animal] models of the long-QT syndrome fully emulate the human disease phenotype.”
This study thus demonstrates “the importance of alternative systems in which human genetic disorders can be studied in the physiologic and disease-causing contexts on a patient-specific level,” the researchers said.
This work was supported by grants from the European Research Council, the German Research Foundation, and the German Ministry for Education and Research. One of the investigators also reported receiving Deutsche Forschungsgemeinschaft grant support.
Copyright (c) 2010 Elsevier Global Medical News. All rights reserved. This material may not be published, broadcast, rewritten, or redistributed.
据10月7日发表于《新英格兰医学杂志》(the New England Journal of Medicine)的一项小规模研究报告，由家族性长QT综合征患者及其亲属的皮肤成纤维细胞诱导生成的多能干细胞，在经进一步诱导分化为心肌细胞后，显现出遗传性心脏病独特的电生理特征。这是研究者首次成功利用干细胞模型重现这种遗传性心脏病。
研究者在对1名8岁男孩进行注意力缺陷多动症(ADHD)临床评估时发现，其心电图QT间期延长，基因分型显示该患者是KSNQ1突变基因携带者(其特征性病变为1型长QT综合征)。研究者获取了该患者及其父亲(携带同一突变基因)的皮肤成纤维细胞，并随后利用这些细胞进行了多能干细胞培养，同时取2名健康受试者的细胞作为对照进行同期平行培养。经诱导后这些细胞分化成了心脏细胞系，在对其施加起搏信号刺激后，研究者据其动作电位差异将这些细胞划分为“心室”、“心房”和“传导束”心肌细胞。
与对照组心肌细胞相比，患者心肌细胞动作电位延长，慢反应延迟整流钾通道(IKs)存在异常活化反应和失活反应，且对儿茶酚胺刺激反应异常，而这些都是长QT综合征的特征性改变。对KSNQ1基因突变进行的进一步分析显示，其特征性“负显性转运缺陷”可导致IKs电流降低70%~80%，还使得支配离子通道激活和失活的各类因子功能发生改变。随后，研究者用异丙肾上腺素对这些心肌细胞施加肾上腺素能刺激，评估发现，对照组心肌细胞动作电位时间缩短，但患者心肌细胞时间延长，这表明，在用药后，这些细胞1型长QT综合征相关表型会表现得更加明显，其心律失常的发病风险也随之升高。利用β受体阻滞剂普萘洛尔对这些细胞进行预处理可以诱导某种保护效应，正如β受体阻滞剂对心室复极相异常延长的患者(此类患者易发生多形性室性心动过速和心源性猝死)有保护效应。
该发现对整个心脏病研究都意义重大，目前研究长QT综合征的发病机制还主要依靠遗传动物模型，但离子通道存在种属间差异，这些模型无法完美模拟人类疾病表型。而这种干细胞模型将为遗传性心脏病新药研发指明一条全新途径，因为其具备多能性且可诱导分化出无限多的心肌细胞，这将为高通量药物研发带来便利，其患者特异性还将为个体化药物研发带来新机遇。
该研究由欧洲研究理事会、德国研究基金会、德国联邦教育研究部赞助。
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