Pálóczi János, Varga Zoltán V, Apáti Ágota, Szebényi Kornélia, Sarkadi Balázs, Madonna Rosalinda, De Caterina Raffaele, Csont Tamás, Eschenhagen Thomas, Ferdinandy Péter, Görbe Anikó
Cardiovascular Research Group, Department of Biochemistry, Faculty of Medicine, University of Szeged, Szeged 6720, Hungary.
Cardiovascular Research Group, Department of Biochemistry, Faculty of Medicine, University of Szeged, Szeged 6720, Hungary; Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest 1085, Hungary.
Oxid Med Cell Longev. 2016;2016:4298945. doi: 10.1155/2016/4298945. Epub 2016 Jun 15.
Background and Aims. Human embryonic stem cell- (hESC-) derived cardiomyocytes are one of the useful screening platforms of potential cardiocytoprotective molecules. However, little is known about the behavior of these cardiomyocytes in simulated ischemia/reperfusion conditions. In this study, we have tested the cytoprotective effect of an NO donor and the brain type natriuretic peptide (BNP) in a screening platform based first on differentiated embryonic bodies (EBs, 6 + 4 days) and then on more differentiated cardiomyocytes (6 + 24 days), both derived from hESCs. Methods. Both types of hESC-derived cells were exposed to 150 min simulated ischemia, followed by 120 min reperfusion. Cell viability was assessed by propidium iodide staining. The following treatments were applied during simulated ischemia in differentiated EBs: the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) (10(-7), 10(-6), and 10(-5) M), BNP (10(-9), 10(-8), and 10(-7) M), and the nonspecific NO synthase inhibitor Nω-nitro-L-arginine (L-NNA, 10(-5) M). Results. SNAP (10(-6), 10(-5) M) significantly attenuated cell death in differentiated EBs. However, simulated ischemia/reperfusion-induced cell death was not affected by BNP or by L-NNA. In separate experiments, SNAP (10(-6) M) also protected hESC-derived cardiomyocytes. Conclusions. We conclude that SNAP, but not BNP, protects differentiated EBs or cardiomyocytes derived from hESCs against simulated ischemia/reperfusion injury. The present screening platform is a useful tool for discovery of cardiocytoprotective molecules and their cellular mechanisms.
背景与目的。人胚胎干细胞（hESC）来源的心肌细胞是潜在心脏细胞保护分子的有用筛选平台之一。然而，对于这些心肌细胞在模拟缺血/再灌注条件下的行为知之甚少。在本研究中，我们在一个筛选平台上测试了一种一氧化氮（NO）供体和脑型利钠肽（BNP）的细胞保护作用，该平台首先基于分化的胚体（EBs，6 + 4天），然后基于更分化的心肌细胞（6 + 24天），二者均来源于hESCs。方法。两种类型的hESC来源细胞均暴露于150分钟的模拟缺血，随后进行120分钟的再灌注。通过碘化丙啶染色评估细胞活力。在分化的EBs模拟缺血期间应用以下处理：NO供体S-亚硝基-N-乙酰青霉胺（SNAP）（10⁻⁷、10⁻⁶和10⁻⁵ M）、BNP（10⁻⁹、10⁻⁸和10⁻⁷ M）以及非特异性NO合酶抑制剂Nω-硝基-L-精氨酸（L-NNA，10⁻⁵ M）。结果。SNAP（10⁻⁶、10⁻⁵ M）显著减轻了分化的EBs中的细胞死亡。然而，模拟缺血/再灌注诱导的细胞死亡不受BNP或L-NNA的影响。在单独的实验中，SNAP（10⁻⁶ M）也保护了hESC来源的心肌细胞。结论。我们得出结论，SNAP而非BNP可保护hESC来源的分化EBs或心肌细胞免受模拟缺血/再灌注损伤。当前的筛选平台是发现心脏细胞保护分子及其细胞机制的有用工具。
