目的: 研究内皮素-1(endothelin, ET-1)对人诱导多能干细胞(human induced pluripotent stem cells, hiPSC)分化形成的人心脏类器官(human heart organoids, hHOs)钙瞬变频率和电生理信号的影响。方法: 进行hHOs的构建和验证,将hiPSC分化为hHOs,并通过形态学和免疫荧光分析进行验证。进行ET-1对hHOs钙瞬变频率及电生理影响的研究。除对照组外,其他各组给予相应浓度的ET-1处理,处理时间分别为0.25 h、1 h、4 h和48 h。处理后,检测hHOs的Ca2+成像和电生理分析。结果: 构建的hHOs实现了单一腔室,均一跳动;培养30 d的hHOs中呈现各种典型心脏细胞类型和组织结构的阳性表达,包括心肌细胞cTnT,心外膜标记物WT1,心室肌细胞MYL2和心房肌细胞MYL7;100 nM的ET-1处理hHOs(0～1 h)无明显影响,而1 μM的ET-1处理同时间段则增加了Ca2+瞬变频率。100 nM和1 μM的ET-1处理(0～8 h)均使hHOs中Ca2+瞬变频率降低,空白对照组在二甲基亚砜(dimethyl sulfoxide,DMSO)暴露8 h内无明显变化;电生理结果显示100 nM和1 μM的ET-1(1～48 h)处理导致搏动频率降低,1 μM的ET-1(0～1 h)处理增加了场电位频率,差异具有统计学意义(P＜0.01)。结论: ET-1可通过调节Ca2+通道及影响心肌细胞收缩功能,对hHOs的钙瞬变频率和电生理信号产生浓度和时间依赖性影响,提示其可能具有心脏毒性及引起病理性重塑风险。
Objective: To explore the effects of endothelin-1 (ET-1) on the calcium transient frequency and electrophysiological signals of human heart organoids (hHOs) induced by human induced pluripotent stem cells (hiPSC). Methods: The construction and verification of hHOs were carried out, and hiPSC was differentiated into hHOs, which was verified by morphology and immunofluorescence analysis. The effects of ET-1 on calcium transient frequency and electrophysiology of hHOs were studied. Except for the control group, the other groups were treated with corresponding concentrations of ET-1 for 0.25 h, 1 h, 4 h and 48 h, respectively. After treatment, Ca2+ imaging and electrophysiological analysis of hHOs were detected. Results: The constructed hHOs achieved a single chamber and uniform beating. The hHOs cultured for 30 days showed positive expression of various typical cardiac cell types and tissue structures, including cTnT, epicardial marker WT1, ventricular myocytes MYL2 and atrial myocytes MYL7. ET-1 (100 nM) had no significant effect on hHOs (0-1 h), while 1 μM ET-1 treatment increased the frequency of Ca2+ transients at the same time. Both 100 nM and 1 μM ET-1 treatment (0-8 h) reduced the transient frequency of Ca2+ in hHOs, and there was no significant change in the blank control group within 8 h of dimethyl sulfoxide (DMSO) exposure. The electrophysiological results showed that 100 nM and 1 μM ET-1 (1-48 h ) treatment led to a decrease in pulse frequency, and 1 μM ET-1 (0-1 h) treatment increased the field potential frequency, the difference was statistically significant (P＜0.01). Conclusion: ET-1 can affect the calcium transient frequency and electrophysiological signal generation of hHOs in a concentration-and time-dependent manner by regulating Ca2+ channels and affecting the contractile function of cardiomyocytes, suggesting that it may have cardiotoxicity and cause pathological remodeling risk.
[1] Xuan W, Tipparaju SM, Ashraf M. Transformational applications of human cardiac organoids in cardiovascular diseases[J]. Frontiers in Cell and Developmental Biology, 2022, 10: 936084.
[2] Cecchetto A, Rampazzo A, Angelini A, et al. From molecular mechanisms of cardiac development to genetic substrate of congenital heart diseases[J]. Future Cardiology, 2010, 6(3): 373-393.
[3] Li Q, Nan K, Le Floch P, et al. Cyborg organoids:implantation of nanoelectronics via organogenesis for tissue-wide electrophysiology[J]. Nano letters, 2019, 19(8): 5781-5789.
[4] Lee J, Sutani A, Kaneko R, et al. In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix[J]. Nature Communications, 2020, 11(1): 4283.
[5] Lewis-Israeli YR, Wasserman AH, Gabalski MA, et al. Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease[J]. Nature Communications, 2021, 12(1): 5142.
[6] Lee SG, Kim YJ, Son MY, et al. Generation of human iPSCs derived heart organoids structurally and functionally similar to heart[J]. Biomaterials, 2022, 290: 121860.
[7] Sabatine MS, Morrow DA, de Lemos JA, et al. Evaluation of multiple biomarkers of cardiovascular stress for risk prediction and guiding medical therapy in patients with stable coronary disease[J]. Circulation, 2012, 125(2): 233-240.
[8] Choisy SC, Cheng H, Smith GL, et al. Modulation by endothelin-1 of spontaneous activity and membrane currents of atrioventricular node myocytes from the rabbit heart[J]. PLoS One, 2012, 7(3): e33448.
[9] Blinova K, Stohlman J, Vicente J et al. Comprehensive translational assessment of human-induced pluripotent stem cell derived cardiomyocytes for evaluating drug-induced arrhythmias[J]. Toxicological Sciences, 2017, 155(1): 234-247.
[10] Kiessling M, Djalinac N, Voglhuber J, et al. Nuclear calcium in cardiac(Patho) Physiology:small compartment, big impact[J]. Biomedicines, 2023, 11(3): 960.
[11] Garcia-Dorado D, Piper HM, Eisner DA. Sarcoplasmic reticulum and mitochondria in cardiac pathophysiology[J]. Cardiovascular Research, 2008, 77(2): 231-233.
[12] Uehara Y, Azuma Y, Minai K, et al. Endothelin-1 prolongs intracellular calcium transient decay in neonatal rat cardiac myocytes[J]. Heart and vessels, 2012, 27: 98-105.
[13] Reisner Y, Meiry G, Zeevi-Levin N, et al. Impulse conduction and gap junctional remodelling by endothelin-1 in cultured neonatal rat ventricular myocytes[J]. Journal of Cellular and Molecular Medicine, 2009, 13(3): 562-573.
[14] Burrell KM, Molenaar P, Dawson PJ, et al. Contractile and arrhythmic effects of endothelin receptor agonists in human heart in vitro: blockade with SB 209670[J]. The Journal of pharmacology and experimental therapeutics, 2000, 292(1): 449-459.
[15] Goldberg AT, Bond BR, Mukherjee R, et al. Endothelin receptor pathway in human left ventricular myocytes:relation to contractility[J]. The Annals of thoracic surgery, 2000, 69(3): 711-715.
[16] Mayyas F, Niebauer M, Zurick A, et al. Association of left atrial endothelin-1 with atrial rhythm, size, and fibrosis in patients with structural heart disease[J]. Circulation: Arrhythmia and Electrophysiology, 2010, 3(4): 369-379.
[17] Wang Y, Tang Y, Zou Y, et al. Plasma level of big endothelin-1 predicts the prognosis in patients with hypertrophic cardiomyopathy[J].International Journal of Cardiology, 2017, 243: 283-289.
[18] Bhandari SS, Davies JE, Struck J, et al. Plasma C-terminal proEndothelin-1 (CTproET-1)is affected by age, renal function, left atrial size and diastolic blood pressure in healthy subjects[J]. Peptides, 2014, 52: 53-57.
[19] Jankowich MD, Wu WC, Choudhary G. Association of elevated plasma endothelin-1 levels with pulmonary hypertension, mortality, and heart failure in African American individuals:the Jackson Heart Study[J]. JAMA Cardiology, 2016, 1(4): 461-469.
