Department of Developmental Cardiology, Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic.
Exp Biol Med (Maywood). 2013 Feb;238(2):233-41. doi: 10.1177/1535370212474596.
The aim of this study was to find out whether opening of mitochondrial large-conductance Ca(2+)-activated potassium channels (BK(Ca)) protects cardiomyocytes against injury caused by simulated ischemia and reperfusion. This study also aimed to determine whether the protective mechanism involves signaling by reactive oxygen species (ROS) and phosphatidylinositol-3-kinase (PI3K). We used isolated ventricular myocytes, which are believed to contain no functional BK(Ca) channels in the sarcolemma. Cells were isolated from the left ventricles of adult male Wistar rats and subjected to 25-min metabolic inhibition with NaCN and 2-deoxyglucose followed by 30-min re-energization. NS11021 (0.1 μmol/L), a novel BK(Ca) channel opener, or hydrogen peroxide (2 μmol/L) added at re-energization, increased cell survival (the number of rod-shaped cells) and markedly reduced the release of lactate dehydrogenase (LDH). These cytoprotective effects of NS11021 were completely abolished by paxilline, a BK(Ca) inhibitor, or tempol, an antioxidant, but not by wortmannin, an inhibitor of PI3K. NS11021 slightly but significantly increased the fluorescence signal in 2'7'-dichlorodihydrofluorescein diacetate (DCF-DA)-loaded myocytes, indicating an increased ROS formation. The NS11021-induced ROS formation was abolished by paxilline or tempol. NS13558 (0.1 μmol/L), an inactive structural analogue of NS11021, affected neither cell survival/LDH release nor DCF-DA fluorescence. These results suggest that pharmacological activation of mitochondrial BK(Ca) channels effectively protects isolated cardiomyocytes against injury associated with simulated reperfusion. The mechanism for this form of protection requires ROS signaling, but not the activation of the PI3K pathway.
本研究旨在探讨线粒体大电导钙激活钾通道（BK(Ca)）的开放是否能保护心肌细胞免受模拟缺血再灌注引起的损伤。本研究还旨在确定保护机制是否涉及活性氧（ROS）和磷脂酰肌醇-3-激酶（PI3K）的信号转导。我们使用分离的心室肌细胞，这些细胞在肌膜中被认为不含有功能的 BK(Ca)通道。细胞从成年雄性 Wistar 大鼠的左心室中分离出来，并在 25 分钟的氰化钠和 2-脱氧葡萄糖代谢抑制后，再进行 30 分钟的再供能。在再供能时加入新型 BK(Ca)通道开放剂 NS11021（0.1 μmol/L）或过氧化氢（2 μmol/L），可增加细胞存活率（杆状细胞的数量），并显著降低乳酸脱氢酶（LDH）的释放。BK(Ca)抑制剂 paxilline或抗氧化剂 tempol 完全消除了 NS11021 的这些细胞保护作用，但 PI3K 抑制剂 wortmannin 则没有。NS11021 可轻微但显著增加 2'7'-二氯二氢荧光素二乙酸酯（DCF-DA）负载的肌细胞中的荧光信号，表明 ROS 形成增加。NS11021 诱导的 ROS 形成被 paxilline 或 tempol 所消除。NS13558（0.1 μmol/L），一种无活性的 NS11021 结构类似物，既不影响细胞存活率/乳酸脱氢酶释放，也不影响 DCF-DA 荧光。这些结果表明，药理学激活线粒体 BK(Ca) 通道可有效保护分离的心肌细胞免受与模拟再灌注相关的损伤。这种保护形式的机制需要 ROS 信号转导，但不需要 PI3K 途径的激活。
