Duchenne肌营养不良症(DMD)心肌病患者有发展危及生命的心律失常的风险,但机制未知.我们旨在确定离子通道控制心脏兴奋性在DMD患者心律失常机制中的作用。
为了测试肌营养不良蛋白突变是否会导致心脏NaV1.5-Kir2.1通道体缺陷和心律失常,我们从两个半合子DMD雄性中产生iPSC-CM,一个杂合的雌性,和两个无关的对照男性。我们进行了包括共聚焦显微镜在内的研究,蛋白质表达分析,贴片夹紧,非病毒piggy-bac基因表达,光学映射和收缩性测定。
两名患者心电图异常,室性心动过速频发。来自所有DMD患者的iPSC-CM显示异常动作电位谱,减慢的传导速度,和减少的钠(INa)和向内整流钾(IK1)电流。膜NaV1.5和Kir2.1蛋白水平在半合子DMDiPSC-CM中降低,但在杂合子iPSC-CM中没有降低。仅在一名患者的半合子iPSC-CM中转染肌营养不良蛋白复合物(α1-syntrophin)的一种成分,可恢复通道体功能,INa和IK1密度,和单个细胞中的动作电位谱。此外,α1-syntrophin表达恢复了hiPSC-CM单层中的冲动传导和收缩力,并防止了折返性心律失常。
我们提供了第一个证明,从患有心肌病的DMD患者的皮肤成纤维细胞重新编程的iPSC-CM具有NaV1.5-Kir2.1通道的功能障碍,从而降低心脏兴奋性和传导。总之,DMD心肌病患者的iPSC-CM具有NaV1.5-Kir2.1通道功能障碍,可以通过支架蛋白α1-synrophin恢复兴奋性并预防心律失常。
由美国国立卫生研究院R01HL122352资助;“laCaixa”银行基金会(HR18-00304);LaMaratóTV3基金会:Ayudasalainvestigaciónenenfermedadesraras2020(LAMARATO-2020);CarrodCarlosIII/FEDER/FSE研究所支持的2020-I65J研究所研究和CienciaeInnovaciónMinisteriodeCienciaeInnovación(MCIN)andtheProCNICFoundation),并且是SeveroOchoa卓越中心(授予CEX2020-001041-S,由MICIN/AEI/10.13039/501100011033资助)。美国心脏协会博士后研究金19POST34380706s至JVEN。以色列OB和MA科学基金会[824/19]。Rappaport赠款[01012020RI];和Niedersachsen基金会[ZN3452]给OB;美国-以色列双边科学基金会(BSF)给OB和TH[2019039];伯纳德·卢布林博士向OB捐款;以及荷兰杜兴父母项目(DPPNL2029771)给OB。美国国立卫生研究院R01AR068428给DM和美国-以色列双边科学基金会资助[2013032]给DM和OB。
Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients.
To test whether dystrophin mutations lead to defective cardiac NaV1.5-Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays.
Two patients had abnormal ECGs with frequent runs of ventricular tachycardia. iPSC-CMs from all DMD patients showed abnormal action potential profiles, slowed conduction velocities, and reduced sodium (INa) and inward rectifier potassium (IK1) currents. Membrane NaV1.5 and Kir2.1 protein levels were reduced in hemizygous DMD iPSC-CMs but not in heterozygous iPSC-CMs. Remarkably, transfecting just one component of the dystrophin protein complex (α1-syntrophin) in hemizygous iPSC-CMs from one patient restored channelosome function, INa and IK1 densities, and action potential profile in single cells. In addition, α1-syntrophin expression restored impulse conduction and contractility and prevented reentrant arrhythmias in hiPSC-CM monolayers.
We provide the first demonstration that iPSC-CMs reprogrammed from skin fibroblasts of DMD patients with cardiomyopathy have a dysfunction of the NaV1.5-Kir2.1 channelosome, with consequent reduction of cardiac excitability and conduction. Altogether, iPSC-CMs from patients with DMD cardiomyopathy have a NaV1.5-Kir2.1 channelosome dysfunction, which can be rescued by the scaffolding protein α1-syntrophin to restore excitability and prevent arrhythmias.
Supported by National Institutes of Health R01 HL122352 grant; \'la Caixa\' Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB.