■人多能干细胞衍生的心肌细胞(hPSC-CM)在心肌梗死(MI)后显示出巨大的心脏再生前景,但是他们的移植在大型动物MI模型中引起短暂性室性心动过速(VT),代表翻译的主要障碍。我们的研究小组先前报道过,这些心律失常起因于移植组织作为异位起搏器的局灶性机制;因此,我们假设,以显性阴性形式的起搏器离子通道HCN4(dnHCN4)工程化的hPSC-CM在移植后表现出降低的自律性和致心律失常风险.
■我们使用CRISPR/Cas9介导的基因编辑来创建转基因dnHCN4hPSC-CM,并通过膜片钳记录和光学作图在体外评估了它们的电生理行为。接下来,我们在猪MI模型中移植了WT和纯合dnHCN4hPSC-CM,并通过免疫组织化学比较了移植后结果,包括自发性心律失常的发生率和移植物结构.
■相对于野生型(WT)心肌细胞,体外dnHCN4hPSC-CM表现出显着降低的自动性和起搏器有趣电流(If)密度。用dnHCN4或WThPSC-CM移植后,所有受者心脏均显示透壁性梗塞瘢痕,该瘢痕被分散的人心肌岛部分再肌肉化。然而,与我们的假设相反,dnHCN4和WThPSC-CM受者均表现出频繁的室性心动过速(VT)发作.
■虽然起搏器离子通道HCN4的遗传沉默在体外抑制了hPSC-CM的自律性,这种干预不足以降低猪MI模型移植后的VT风险,这意味着更复杂的机制在体内是有效的。
UNASSIGNED: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show tremendous promise for cardiac regeneration following myocardial infarction (MI), but their transplantation gives rise to transient ventricular tachycardia (VT) in large-animal MI models, representing a major hurdle to translation. Our group previously reported that these arrhythmias arise from a focal mechanism whereby graft tissue functions as an ectopic pacemaker; therefore, we hypothesized that hPSC-CMs engineered with a dominant negative form of the pacemaker ion channel HCN4 (dnHCN4) would exhibit reduced automaticity and arrhythmogenic risk following transplantation.
UNASSIGNED: We used CRISPR/Cas9-mediated gene-editing to create transgenic dnHCN4 hPSC-CMs, and their electrophysiological behavior was evaluated in vitro by patch-clamp recordings and optical mapping. Next, we transplanted WT and homozygous dnHCN4 hPSC-CMs in a pig MI model and compared post-transplantation outcomes including the incidence of spontaneous arrhythmias and graft structure by immunohistochemistry.
UNASSIGNED: In vitro dnHCN4 hPSC-CMs exhibited significantly reduced automaticity and pacemaker funny current (I f ) density relative to wildtype (WT) cardiomyocytes. Following transplantation with either dnHCN4 or WT hPSC-CMs, all recipient hearts showed transmural infarct scar that was partially remuscularized by scattered islands of human myocardium. However, in contrast to our hypothesis, both dnHCN4 and WT hPSC-CM recipients exhibited frequent episodes of ventricular tachycardia (VT).
UNASSIGNED: While genetic silencing of the pacemaker ion channel HCN4 suppresses the automaticity of hPSC-CMs in vitro, this intervention is insufficient to reduce VT risk post-transplantation in the pig MI model, implying more complex mechanism(s) are operational in vivo.