Kim Jong J, Yang Lei, Lin Bo, Zhu Xiaodong, Sun Bin, Kaplan Aaron D, Bett Glenna C L, Rasmusson Randall L, London Barry, Salama Guy
Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261, USA; Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
J Mol Cell Cardiol. 2015 Apr;81:81-93. doi: 10.1016/j.yjmcc.2015.01.013. Epub 2015 Jan 30.
The creation of cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs) has spawned broad excitement borne out of the prospects to diagnose and treat cardiovascular diseases based on personalized medicine. A common feature of hiPS-CMs is their spontaneous contractions but the mechanism(s) remain uncertain.
Intrinsic activity was investigated by the voltage-clamp technique, optical mapping of action potentials (APs) and intracellular Ca(2+) (Cai) transients (CaiT) at subcellular-resolution and pharmacological interventions.
The frequency of spontaneous CaiT (sCaiT) in monolayers of hiPS-CMs was not altered by ivabradine, an inhibitor of the pacemaker current, If despite high levels of HCN transcripts (1-4). HiPS-CMs had negligible If and IK1 (inwardly-rectifying K(+)-current) and a minimum diastolic potential of -59.1±3.3mV (n=18). APs upstrokes were preceded by a depolarizing-foot coincident with a rise of Cai. Subcellular Cai wavelets varied in amplitude, propagated and died-off; larger Cai-waves triggered cellular sCaTs and APs. SCaiTs increased in frequency with [Ca(2+)]out (0.05-to-1.8mM), isoproterenol (1μM) or caffeine (100μM) (n≥5, p<0.05). HiPS-CMs became quiescent with ryanodine receptor stabilizers (K201=2μM); tetracaine; Na-Ca exchange (NCX) inhibition (SEA0400=2μM); higher [K(+)]out (5→8mM), and thiol-reducing agents but could still be electrically stimulated to elicit CaiTs. Cell-cell coupling of hiPS-CM in monolayers was evident from connexin-43 expression and CaiT propagation. SCaiTs from an ensemble of dispersed hiPS-CMs were out-of-phase but became synchronous through the outgrowth of inter-connecting microtubules.
Automaticity in hiPS-CMs originates from a Ca(2+)-clock mechanism involving Ca(2+) cycling across the sarcoplasmic reticulum linked to NCX to trigger APs.
源自人诱导多能干细胞（hiPS-CMs）的心肌细胞的产生引发了广泛的关注，这源于基于个性化医疗来诊断和治疗心血管疾病的前景。hiPS-CMs的一个共同特征是它们的自发收缩，但机制仍不明确。
通过电压钳技术、亚细胞分辨率下动作电位（APs）和细胞内Ca(2+)（Cai）瞬变（CaiT）的光学映射以及药理学干预来研究内在活性。
尽管HCN转录本（1-4）水平较高，但起搏电流If的抑制剂伊伐布雷定并未改变hiPS-CMs单层中自发CaiT（sCaiT）的频率。hiPS-CMs的If和IK1（内向整流K(+)电流）可忽略不计，最小舒张电位为-59.1±3.3mV（n = 18）。APs的上升支之前有一个与Cai升高同时出现的去极化脚。亚细胞Cai小波的振幅各不相同，传播并消失；较大的Cai波触发细胞sCaTs和APs。随着细胞外[Ca(2+)]（0.05至1.8mM）、异丙肾上腺素（1μM）或咖啡因（100μM）浓度升高，sCaiTs的频率增加（n≥5，p<0.05）。hiPS-CMs在使用兰尼碱受体稳定剂（K201 = 2μM）、丁卡因、钠钙交换（NCX）抑制（SEA0400 = 2μM）、较高的细胞外[K(+)]（5→8mM）和硫醇还原剂时会静止，但仍可被电刺激引发CaiTs。从连接蛋白-43表达和CaiT传播可明显看出hiPS-CMs单层中的细胞间偶联。分散的hiPS-CMs群体的sCaiTs不同步，但通过相互连接的微管生长变得同步。
hiPS-CMs的自律性源于一种Ca(2+)时钟机制，该机制涉及Ca(2+)在肌浆网中的循环，与NCX相连以触发APs。
