Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany.
Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Department of Cardiology, University Heart and Vascular Center, Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany.
Cell Signal. 2021 Jun;82:109970. doi: 10.1016/j.cellsig.2021.109970. Epub 2021 Mar 5.
There is ongoing interest in generating cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) to study human cardiac physiology and pathophysiology. Recently we found that norepinephrine-stimulated calcium currents (I) in hiPSC-cardiomyocytes were smaller in conventional monolayers (ML) than in engineered heart tissue (EHT). In order to elucidate culture specific regulation of β-adrenoceptor (β-AR) responses we investigated whether action of phosphodiesterases (PDEs) may limit norepinephrine effects on I and on cytosolic cAMP in hiPSC-cardiomyocytes. Results were compared to adult human atrial cardiomyocytes.
Adult human atrial cardiomyocytes were isolated from tissue samples obtained during open heart surgery. All patients were in sinus rhythm. HiPSC-cardiomyocytes were dissociated from ML and EHT. Förster-resonance energy transfer (FRET) was used to monitor cytosolic cAMP (Epac1-camps sensor, transfected by adenovirus). I was recorded by whole-cell patch clamp technique. Cilostamide (300 nM) and rolipram (10 μM) were used to inhibit PDE3 and PDE4, respectively. β-AR were stimulated with the physiological agonist norepinephrine (100 μM).
In adult human atrial cardiomyocytes, norepinephrine increased cytosolic cAMP FRET ratio by +13.7 ± 1.5% (n = 10/9, mean ± SEM, number of cells/number patients) and I by +10.4 ± 1.5 pA/pF (n = 15/10). This effect was not further increased in the concomitant presence of rolipram, cilostamide and norepinephrine, indicating saturation by norepinephrine alone. In ML hiPSC-cardiomyocytes, norepinephrine exerted smaller increases in cytosolic cAMP and I (FRET +9.6 ± 0.5% n = 52/21, number of cells/number of ML or EHT, and I + 1.4 ± 0.2 pA/pF n = 34/7, p < 0.05 each) and both were augmented in the presence of the PDE4 inhibitor rolipram (FRET +16.7 ± 0.8% n = 94/26 and I + 5.6 ± 1.4 pA/pF n = 11/5, p < 0.05 each). Cilostamide increased the response to norepinephrine on FRET (+12.7 ± 0.5% n = 91/19, p < 0.05), but not on I. In EHT hiPSC-cardiomyocytes, norepinephrine responses on both, FRET and I, were larger than in ML (FRET +12.1 ± 0.3% n = 87/32 and I + 3.3 ± 0.2 pA/pF n = 13/5, p < 0.05 each). Rolipram augmented the norepinephrine effect on I (+6.2 ± 1.6 pA/pF; p < 0.05 vs. norepinephrine alone, n = 10/4), but not on FRET.
Our results show culture-dependent differences in hiPSC-cardiomyocytes. In conventional ML but not in EHT, maximum norepinephrine effects on cytosolic cAMP depend on PDE3 and PDE4, suggesting immaturity when compared to the situation in adult human atrial cardiomyocytes. The smaller I responses to norepinephrine in ML and EHT vs. adult human atrial cardiomyocytes depend at least partially on a non-physiological large impact of PDE4 in hiPSC-cardiomyocytes.
人们对利用人诱导多能干细胞（hiPSC）生成心肌细胞以研究人类心脏生理学和病理生理学一直很感兴趣。最近我们发现，与工程化心脏组织（EHT）相比，hiPSC 心肌细胞在常规单层（ML）中的去甲肾上腺素刺激钙电流（I）较小。为了阐明培养特异性对β-肾上腺素能受体（β-AR）反应的调节，我们研究了磷酸二酯酶（PDEs）的作用是否可能限制去甲肾上腺素对 I 和 hiPSC 心肌细胞胞质 cAMP 的影响。结果与成人人心房肌细胞进行了比较。
从心脏直视手术中获得的组织样本中分离出成人人心房肌细胞。所有患者均处于窦性心律。hiPSC 心肌细胞从 ML 和 EHT 中分离出来。通过荧光共振能量转移（FRET）监测胞质 cAMP（通过腺病毒转染的 Epac1-camps 传感器）。通过全细胞膜片钳技术记录 I。使用西洛酰胺（300 nM）和罗利普兰（10 μM）分别抑制 PDE3 和 PDE4。用生理激动剂去甲肾上腺素（100 μM）刺激β-AR。
在成人人心房肌细胞中，去甲肾上腺素使胞质 cAMP FRET 比率增加+13.7±1.5%（n=10/9，均值±SEM，细胞数/患者数），I 增加+10.4±1.5 pA/pF（n=15/10）。在罗利普兰、西洛酰胺和去甲肾上腺素同时存在的情况下，这种作用没有进一步增加，表明仅由去甲肾上腺素引起的饱和。在 ML hiPSC 心肌细胞中，去甲肾上腺素对胞质 cAMP 和 I 的增加较小（FRET+9.6±0.5%，n=52/21，ML 或 EHT 的细胞数/细胞数，I+1.4±0.2 pA/pF，n=34/7，p<0.05 各），并且在 PDE4 抑制剂罗利普兰存在下均增加（FRET+16.7±0.8%，n=94/26 和 I+5.6±1.4 pA/pF，n=11/5，p<0.05 各）。西洛酰胺增加了去甲肾上腺素对 FRET 的反应（+12.7±0.5%，n=91/19，p<0.05），但对 I 没有影响。在 EHT hiPSC 心肌细胞中，FRET 和 I 上的去甲肾上腺素反应均大于 ML（FRET+12.1±0.3%，n=87/32 和 I+3.3±0.2 pA/pF，n=13/5，p<0.05 各）。罗利普兰增强了去甲肾上腺素对 I 的作用（+6.2±1.6 pA/pF；与去甲肾上腺素单独作用相比，p<0.05，n=10/4），但对 FRET 没有影响。
我们的结果显示 hiPSC 心肌细胞存在培养依赖性差异。在常规 ML 中，但不在 EHT 中，去甲肾上腺素对胞质 cAMP 的最大作用依赖于 PDE3 和 PDE4，与成人人心房肌细胞相比，提示不成熟。与成人人心房肌细胞相比，ML 和 EHT 中去甲肾上腺素对 I 的反应较小至少部分归因于 hiPSC 心肌细胞中 PDE4 的非生理大影响。
