Uzun Ahmet U, Mannhardt Ingra, Breckwoldt Kaja, Horváth András, Johannsen Silke S, Hansen Arne, Eschenhagen Thomas, Christ Torsten
Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-EppendorfHamburg, Germany; Partner Site Hamburg/Kiel/Lübeck, German Centre for Cardiovascular Research (DZHK)Hamburg, Germany.
Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-EppendorfHamburg, Germany; Partner Site Hamburg/Kiel/Lübeck, German Centre for Cardiovascular Research (DZHK)Hamburg, Germany; Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of SzegedSzeged, Hungary.
Front Pharmacol. 2016 Sep 12;7:300. doi: 10.3389/fphar.2016.00300. eCollection 2016.
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) provide a unique opportunity to study human heart physiology and pharmacology and repair injured hearts. The suitability of hiPSC-CM critically depends on how closely they share physiological properties of human adult cardiomyocytes (CM). Here we investigated whether a 3D engineered heart tissue (EHT) culture format favors maturation and addressed the L-type Ca(2+)-current (ICa,L) as a readout. The results were compared with hiPSC-CM cultured in conventional monolayer (ML) and to our previous data from human adult atrial and ventricular CM obtained when identical patch-clamp protocols were used. HiPSC-CM were two- to three-fold smaller than adult CM, independently of culture format [capacitance ML 45 ± 1 pF (n = 289), EHT 45 ± 1 pF (n = 460), atrial CM 87 ± 3 pF (n = 196), ventricular CM 126 ± 8 pF (n = 50)]. Only 88% of ML cells showed ICa, but all EHT. Basal ICa density was 10 ± 1 pA/pF (n = 207) for ML and 12 ± 1 pA/pF (n = 361) for EHT and was larger than in adult CM [7 ± 1 pA/pF (p < 0.05, n = 196) for atrial CM and 6 ± 1 pA/pF (p < 0.05, n = 47) for ventricular CM]. However, ML and EHT showed robust T-type Ca(2+)-currents (ICa,T). While (-)-Bay K 8644, that activates ICa,L directly, increased ICa,Lto the same extent in ML and EHT, β1- and β2-adrenoceptor effects were marginal in ML, but of same size as (-)-Bay K 8644 in EHT. The opposite was true for serotonin receptors. Sensitivity to β1 and β2-adrenoceptor stimulation was the same in EHT as in adult CM (-logEC50: 5.9 and 6.1 for norepinephrine (NE) and epinephrine (Epi), respectively), but very low concentrations of Rp-8-Br-cAMPS were sufficient to suppress effects (-logEC50: 5.3 and 5.3 respectively for NE and Epi). Taken together, hiPSC-CM express ICa,L at the same density as human adult CM, but, in contrast, possess robust ICa,T. Increased effects of catecholamines in EHT suggest more efficient maturation.
人诱导多能干细胞衍生的心肌细胞（hiPSC-CM）为研究人类心脏生理学、药理学以及修复受损心脏提供了独特的机会。hiPSC-CM的适用性关键取决于它们与人类成年心肌细胞（CM）生理特性的相似程度。在此，我们研究了三维工程心脏组织（EHT）培养形式是否有利于细胞成熟，并将L型钙电流（ICa,L）作为一个衡量指标。将结果与在传统单层（ML）培养的hiPSC-CM进行比较，并与我们之前使用相同膜片钳方案从人类成年心房和心室CM获得的数据进行比较。hiPSC-CM比成年CM小两到三倍，与培养形式无关[ML电容45±1 pF（n = 289），EHT电容45±1 pF（n = 460），心房CM电容87±3 pF（n = 196），心室CM电容126±8 pF（n = 50）]。ML细胞中只有88%显示出ICa,L，但所有EHT细胞均显示有该电流。ML的基础ICa密度为10±1 pA/pF（n = 207），EHT为12±1 pA/pF（n = 361），且大于成年CM[心房CM为7±1 pA/pF（p < 0.05，n = 196），心室CM为6±1 pA/pF（p < 0.05，n = 47）]。然而，ML和EHT均显示出较强的T型钙电流（ICa,T）。虽然直接激活ICa,L的（-）-Bay K 8644在ML和EHT中使ICa,L增加的程度相同，但β1和β2肾上腺素能受体效应在ML中很微弱，而在EHT中与（-）-Bay K 8644的效应大小相同。血清素受体的情况则相反。EHT中对β1和β2肾上腺素能受体刺激的敏感性与成年CM相同（去甲肾上腺素（NE）和肾上腺素（Epi）的-logEC50分别为5.9和6.1），但极低浓度的Rp-8-Br-cAMPS就足以抑制其效应（NE和Epi的-logEC50分别为5.3和5.3）。综上所述，hiPSC-CM表达的ICa,L密度与人类成年CM相同，但与之相反的是，hiPSC-CM具有较强的ICa,T。儿茶酚胺在EHT中的效应增强表明其成熟效率更高。
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